JP5526352B2 - Cell culture conditioned medium composition and method of use - Google Patents

Abstract

Novel products comprising conditioned cell culture medium compositions and methods of use are described. The conditioned cell medium compositions of the invention may be comprised of any known defined or undefined medium and may be conditioned using any eukaryotic cell type. Once the cell medium of the invention is conditioned, it may be used in any state. Physical embodiments of the conditioned medium include, but are not limited to, liquid or solid, frozen, lyophilized or dried into a powder. Additionally, the medium is formulated with a pharmaceutically acceptable carrier as a vehicle for internal administration, applied directly to a food item or product, or formulated with a salve or ointment for topical applications. Also, the medium may be further processed to concentrate or reduce one or more factors or components contained within the medium.

Description

1. INTRODUCTION The present invention relates to a composition comprising a cell culture medium conditioned by cells grown in two-dimensional cultures (ie, monolayers) or three-dimensional cultures. By genetically modifying the cells used to conditioned the medium, the concentration of protein present in the medium can be altered. Cell conditioned media is treated according to a variety of uses, including wound preparations, cosmetic additives, nutritional supplements, animal feed nutritional supplements, cultured cells, pharmaceutical applications, and compositions and methods for promoting hair growth Is done. The present invention also relates to compositions comprising extracellular matrix proteins and / or other purified proteins derived from conditioned media.

2. Background of the Invention
2.1. Cell conditioned media compositions typically contain essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids and nucleosides. The purpose of the cell culture medium is to supply the ingredients necessary to meet the nutritional requirements required to grow the cells in a controlled artificial and in vitro environment. Nutrient composition, pH, and osmolarity vary depending on parameters such as cell type, cell density, and culture system used. Many cell culture media formulations are described in the literature and many media are commercially available. It is known to those skilled in the art that once the medium is incubated with the cells, the medium is referred to as “post-use medium” or “conditioned medium”. The conditioned medium contains various cellular metabolites and secreted proteins such as, for example, bioactive growth factors, inflammatory mediators, and other extracellular proteins, along with most of the initial components of the medium. In contrast to cells grown in three dimensions, cell lines grown as monolayers or on beads are characterized by cell-cell and cell-matrix interactions that are characteristic of the whole tissue in vivo. Lack of action. As a result, although such cells secrete various cellular metabolites, they do not necessarily secrete these metabolites and secreted proteins at levels close to physiological levels. Conventional cell conditioned media, ie, media in which cell lines grown as monolayers or on beads are usually discarded or, in some cases, used for culture operations such as reducing cell density Is done.

2.2. The majority of in vitro cell cultures of tissue culture vertebrates grow as monolayers on artificial substrates soaked in the medium. The nature of the substrate on which the monolayer is grown may be a solid such as plastic or a semi-solid gel such as collagen or agar. Recently, disposable plastics have become the preferred substrate for use in tissue or cell culture.

  Several researchers have studied the use of natural substrates related to basement membrane components. The basement membrane contains a mixture of glycoproteins and proteoglycans that surround most cells in vivo. See, for example, Reid and Rojkund, 1979, Methods in Enzymology, Vol. 57, Cell Culture, Jakoby & Pasten (eds.), New York, Acad, Press, pp. 263-278; Vlodavsky et al., 1980, Cell 19: 607- 617; Yang et al., 1979, Proc. Natl. Acad. Sci. USA 76: 3401 uses collagen to culture hepatocytes, epithelial cells and endothelial tissue. Using cell growth on floating collagen (Michalopoulos and Pitot, 1975, Fed. Proc. 34: 826) and cellulose nitrate membrane (Savage and Bonney, 1978, Exp. Cell Res. 114: 307-315) Attempts have been made to promote differentiation. However, long-term cell regeneration and culture of the tissue in such a culture system has not been achieved so far.

  Mouse embryo fibroblast cultures have been used to enhance cell growth, particularly at low densities. This effect may be due in part to media supplementation, but may also be due to substrate acclimation by cell products. In these systems, the fibroblast feeder cell layer grows as a confluent monolayer, which makes the surface suitable for the attachment of other cells. For example, glioma growth on a confluent support layer of normal fetal intestine has been reported (Lindsay, 1979, Nature 228: 80).

  Two-dimensional cell growth is a preferred method for preparing, observing, and studying cells in culture and allows for rapid cell growth but lacks the characteristics of whole tissue in vivo . In order to study such functional and morphological interactions, several researchers have worked on collagen gels (Douglas et al., 1980, In Vitro 16: 306-312; Yang et al., 1979, Proc. Natl. Acad Sci. 76: 3401; Yang et al., 1980, Proc. Natl. Acad. Sci. 77: 2088-2092; Yang et al., 1981, Cancer Res. 41: 1021-1027); cellulose sponge, alone (Leighton et al., 1951, J. Natl. Cancer Inst. 12: 545-561) or collagen coated (Leighton et al., 1968, Cancer Res. 28: 286-296); gelatin sponge, gel foam (Sorour et al., 1975, J. Neurosurg. 43: 742-749) and other three-dimensional substrates are being studied.

  Usually, these three-dimensional substrates are inoculated with cells to be cultured. Many types of cells have been reported to invade the matrix and establish “tissue-like” tissues. For example, breast epithelium (Yang et al., 1981, Cancer Res. 41: 1021-1027) and sympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98: 159-169) are cultured using a three-dimensional collagen gel. Yes. In addition, various attempts have been made to regenerate tissue-like structures from dispersed monolayer cultures. Kruse and Miedema (1965, J. Cell Biol. 27: 273) report that perfusion monolayers can grow to depths of more than 10 cells, and if you continue to supply the appropriate media, multi-layer culture Can form organ-like structures (Schneider et al., 1963, Exp. Cell. Res. 30: 449-459; Bell et al., 1979, Proc. Natl. Acad. Sci. USA 76: 1274-1279 See also Green, 1978, Science 200: 1385-1388). It has been reported that keratinocytes in the human epidermis can form dematoglyphs (friction bumps that develop when maintained for several weeks without transfer), which are described by Folkman and Haudenschild (1980). , Nature 288: 551-556) report that capillaries are formed in cultures of vascular endothelial cells cultured in the presence of endothelial growth factor and medium conditioned by tumor cells; Sirica et al. (1979, Proc. Natl. Acad. Sci. USA 76: 283-287; 1980, Cancer Res. 40: 3259-3267) on a nylon mesh coated with a thin collagen layer for about 10-13 days. Hepatocytes were maintained in primary culture. However, long-term culture and growth of cells in such a culture system has not been achieved.

  Attempts have been made to establish long-term culture of tissues such as bone marrow. Interstitial cell layers containing various types of cells are rapidly formed, but the overall results were not satisfactory because significant hematopoiesis could not be maintained in real time (as a review, (See Dexter et al., In Long Term Bone Marrow Culture, 1984, Alan R. Liss, Inc., pp. 57-96).

  Numerous groups have attempted to grow skin and connective tissue in vitro for transplantation in vivo. In one such system, a hydrated bovine collagen lattice forms a matrix that incorporates cells such as fibroblasts, causing contraction of the lattice to tissue (Bell et al., US Pat. No. 4,485,096). In another system, fibroblasts are cultured using a porous cross-linked collagen sponge (Eisenberg, WO 91/16010). In addition, it is described that a scaffold composed of a synthetic polymer controls cell growth and proliferation in vitro, so that once fibroblasts proliferate and begin to attach to the matrix, they can be transplanted into a subject ( Vacanti et al., US Pat. Nos. 5,759,830; 5,770,193; 5,736,372).

  Synthetic matrices composed of polyester biodegradable, biocompatible copolymers and amino acids have also been designed as scaffolds for cell growth (US Pat. Nos. 5,654,381; 5,709,854). Similarly, non-biodegradable scaffolds can support cell growth. Three-dimensional cell culture systems have also been designed that consist of a stromal matrix that supports the growth of cells from any desired tissue to adult tissue (Naughton et al., US Pat. Nos. 4,721,096 and 5,032,508). Another approach uses a hydrogel that contains a large number of desired cell types and gradually polymerizes and solidifies into a matrix when administered to a subject (US Pat. No. 5,709,854). An extracellular matrix preparation composed of stromal cells has also been designed, which provides a three-dimensional cell culture system for the desired type of cells, which can be applied at the exact location of the biomaterial. It can be injected into a rug (Naughton et al., WO 96/39101).

2.3. Secretion of extracellular proteins, such as cellular cytokines and growth factors, growth factors, cytokines, and stress proteins, into cell conditioned media can lead to tissue repair (eg wounds and other tissue defects such as cosmetic defects). It opens up new possibilities for the production of products used in a very wide area, including those in therapy) and in human and animal feed supplements. For example, growth factors are known to play an important role in the wound healing process. In general, in the treatment of wounds, it is considered desirable to enhance the supply of growth factors by the direct addition of growth factors.

  Cellular cytokines and growth factors are involved in many important cellular processes including cell proliferation, cell adhesion, cell morphological appearance, differentiation, migration, inflammatory response, angiogenesis, and cell death. Studies have shown that hypoxic stress and damage to cells can affect growth factors such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor). It has been shown to induce responses including increased levels of corresponding mRNA and protein (Gonzalez-Rubio, M et al., 1996, Kidney Int, 50 (1): 164-73; Abramovitch, R. et al., 1997, Int J. Exp. Pathol. 78 (2): 57-70; Stein, I. et al., 1995, Mol Cell Biol. 15 (10): 5363-8; Yang, W. et al., 1997, FEBS Lett. 403 ( 2): 139-42; West, NR et al., 1995, J. Neurosci. Res. 40 (5): 647-59).

  Growth factors such as transforming growth factor-β, also known in the art as TGF-β, are induced by specific stress proteins during wound healing. Two known stress proteins are GRP78 and HSP90. These proteins stabilize the cell structure and make the cells resistant to adverse conditions. The TGF-β family of dimeric proteins includes TGF-β1, TGF-β2 and TGF-β3 and regulates the growth and differentiation of many cell types. In addition, this family of proteins exerts certain biological effects and stimulates the growth of one type of cell (Noda et al., 1989, Endocrinology 124: 2991-2995) or the growth of another type of cell. (Goey et al., 1989, J. Immunol. 143: 877-880; Pietenpol et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3758-3762). TGF-β also increases the expression of extracellular matrix proteins such as collagen and fibronectin (Ignotz et al., 1986, J. Biol. Chem. 261: 4337-4345) and accelerates wound healing (Mustoe et al., 1987, Science 237: 1333-1335).

  PDGF is another growth factor. PDGF was first found to be a potent mitogen for cells derived from mesenchyme (Ross R. et al., 1974, Proc. Natl. Acad. Sci. USA 71 (4): 1207-1210; Kohler N. et al., 1974, Exp. Cell Res. 87: 297-301). Further studies have shown that PDGF increases cell integrity and the rate of granulation in tissue formation. Wounds treated with PDGF develop an early stage inflammatory response including an increase in neutrophil and macrophage cell types at the wound site. In these wounds, enhanced fibroblast function is also observed (Pierce, G. F. et al., 1988, J. Exp. Med. 167: 974-987). Both PDGF and TGF-β have been shown to increase collagen formation, DNA content, and protein levels in animal studies (Grotendorst, GR et al., 1985, J. Clin. Invest. 76: 2323-2329; Sporn, MB et al., 1983, Science (Wash DC) 219: 1329). PDGF has been found to be effective in treating human wounds. In human wounds, PDGF-AA expression is increased in pressure ulcers during healing. Increased PDGF-AA leads to increased activated fibroblasts in the wound, extracellular matrix deposition, and active angiogenesis. Furthermore, such an increase in PDGF-AA is not seen in chronic non-healing wounds (Principles of Tissue Engineering, R. Lanza et al. (Ed.), Pp. 133-141 (RG Landes Co. TX 1997)). A number of other growth factors capable of inducing angiogenesis and wound healing include VEGF, KGF and basic FGF.

  There is currently no simply effective method or composition for applications involving various cytokines, growth factors or other regulatory proteins found in our conditioned media.

U.S. Pat.No. 4,485,096 International Publication No. 91/16010 Pamphlet U.S. Pat.No. 5,759,830 U.S. Pat.No. 5,770,193 U.S. Pat.No. 5,736,372 U.S. Pat.No. 5,654,381 U.S. Pat.No. 5,709,854 U.S. Pat.No. 4,721,096 U.S. Pat.No. 5,032,508 U.S. Patent No. 5,709,854 International Publication No. 96/39101 Pamphlet

Reid and Rojkund, 1979, Methods in Enzymology, Vol. 57, Cell Culture, Jakoby & Pasten (ed.), New York, Acad, Press, pp. 263-278; Vlodavsky et al., 1980, Cell 19: 607-617; Yang et al., 1979, Proc. Natl. Acad. Sci. USA 76: 3401 Michalopoulos and Pitot, 1975, Fed. Proc. 34: 826) Savage and Bonney, 1978, Exp.Cell Res. 114: 307-315 Lindsay, 1979, Nature 228: 80 Douglas et al., 1980, In Vitro 16: 306-312 Yang et al., 1979, Proc. Natl. Acad. Sci. 76: 3401 Yang et al., 1980, Proc. Natl. Acad. Sci. 77: 2088-2092 Yang et al., 1981, Cancer Res. 41: 1021-1027) Leighton et al., 1951, J. Natl. Cancer Inst. 12: 545-561 Leighton et al., 1968, Cancer Res. 28: 286-296 Sorour et al., 1975, J. Neurosurg. 43: 742-749 Yang et al., 1981, Cancer Res. 41: 1021-1027 Ebendal, 1976, Exp.Cell Res.98: 159-169 Kruse and Miedema (1965, J. Cell Biol. 27: 273 Schneider et al., 1963, Exp. Cell. Res. 30: 449-459 Bell et al., 1979, Proc. Natl. Acad. Sci. USA 76: 1274-1279 Green, 1978, Science 200: 1385-1388 Folkman and Haudenschild (1980, Nature 288: 551-556 Sirica et al., 1979, Proc. Natl. Acad. Sci. USA 76: 283-287 1980, Cancer Res. 40: 3259-3267 Dexter et al., In Long Term Bone Marrow Culture, 1984, Alan R. Liss, Inc., pp. 57-96 Gonzalez-Rubio, M et al., 1996, Kidney Int, 50 (1): 164-73 Abramovitch, R. et al., 1997, Int J. Exp. Pathol. 78 (2): 57-70 Stein, I. et al., 1995, Mol Cell Biol. 15 (10): 5363-8 Yang, W. et al., 1997, FEBS Lett. 403 (2): 139-42 West, N.R., et al., 1995, J. Neurosci. Res. 40 (5): 647-59 Noda et al., 1989, Endocrinology 124: 2991-2995 Goey et al., 1989, J. Immunol. 143: 877-880 Pietenpol et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3758-3762 Ignotz et al., 1986, J. Biol. Chem. 261: 4337-4345 Mustoe et al., 1987, Science 237: 1333-1335 Ross R. et al., 1974, Proc. Natl. Acad. Sci. USA 71 (4): 1207-1210 Kohler N. et al., 1974, Exp. Cell Res. 87: 297-301 Pierce, G. F. et al., 1988, J. Exp. Med. 167: 974-987 Grotendorst, G.R., et al., 1985, J. Clin.Invest. 76: 2323-2329 Sporn, M.B., et al., 1983, Science (Wash DC) 219: 1329 Principles of Tissue Engineering, R. Lanza et al. (Ed.), Pp. 133-141, R.G. Landes Co. TX 1997

3. SUMMARY OF THE INVENTION The inventors of the present invention have discovered a novel cell culture conditioned medium composition. In addition, the present invention encompasses the use of such novel compositions. The present invention further includes compositions containing specific protein products derived from the cell conditioned media of the present invention.

  The composition of the cell conditioned medium of the present invention may be composed of any of the known defined or undefined media and may be conditioned using any kind of eukaryotic cells. It can be conditioned with stromal cells, parenchymal cells, mesenchymal cells, liver replacement cells, neural stem cells, pancreatic stem cells, and / or embryonic stem cells. A three-dimensional tissue construct is preferred. Whether monolayer or three-dimensional, the cell type determines the properties of the conditioned medium. For example, conditioned media with astrocytes and nerve cells contain certain characteristic metabolites and proteins, so that conditioned media is preferred for neurorepair applications. In a preferred embodiment, the media of the invention is conditioned with three-dimensional cell and tissue culture. In another preferred embodiment, it is conditioned with stromal cells used for the production of TransCyte ™ (Smith & Nephew PLC., UK). In a highly preferred embodiment, the cells of the three-dimensional tissue culture cells are stromal cells and the tissue culture construct is Dermagraft® (Advanced Tissue Sciences, with or without specific parenchymal cells added). Inc., La Jolla CA). Such cell conditioned media provides a unique combination of factors and their unique ratios, which are different from those of monolayer cultures and are closer to those found in vivo. Three-dimensional stromal culture may be further cultured in parenchymal cells such as epidermis, bone, liver, nerves, pancreas, etc., thereby containing characteristic extracellular proteins and other metabolites of that tissue type A conditioned medium is obtained. Furthermore, each cell type may generally be modified. By genetic modification, the concentration of one or more components in the medium can be changed, for example, for the purpose of up-regulating proteins, introducing new proteins, or controlling ion concentrations.

  The cell culture medium of the present invention may be used in any situation once conditioned. The physical embodiment of the conditioned medium may be, but is not limited to, liquid or solid, frozen body, lyophilized or dried powder. Furthermore, the medium may be formulated with a pharmaceutically acceptable carrier as a vehicle for in vivo administration, may be applied directly as a food material or edible product, The ointment can be formulated, or it can be added during the manufacture of a surgical adhesive, for example to promote the healing of sutures after an invasive procedure, or can be added to the adhesive. Further, the medium can be further treated to concentrate or reduce one or more factors or components contained in the medium. For example, the conditioned medium can be enriched with growth factors using immunoaffinity chromatography.

  In certain embodiments, the conditioned media of the present invention can also be used for wound healing. As an example, the cell conditioned medium can be applied to bandage (adhesive or non-adhesive) gauze and used for topical application to promote and / or accelerate wound healing. The conditioned medium can also be treated to concentrate or reduce one or more components that promote wound healing. The ingredients can be cold dried / freeze dried and added as a wound filler or added to an existing wound filling ingredient to speed wound healing. Alternatively, the medium can be added to the hydrogel composition and used as a film and anti-adhesive agent for topical wound treatment. The medium composition of the present invention may be acclimatized with cells that express a gene product with improved wound healing properties, ie, genetically engineered cells that express a gene product with anti-scarring properties.

  In other embodiments, cell conditioned media formulations of the invention can be used to correct congenital abnormalities or acquired physical defects. In addition, it can be formulated for injection or in the form of a hydrogel and used to restore wrinkles, eyebrows, scar disappearance and other skin conditions. In other embodiments, the cell conditioned medium can be added to eye shadows, cosmetic pancakes, compacts or other cosmetics.

  In yet another embodiment, the cell conditioned medium formulation of the present invention is used as a food additive or nutritional supplement. The conditioned medium contains a plurality of nutrient components such as essential amino acids, vitamins and minerals. The cell conditioned medium of the present invention can be concentrated and / or lyophilized, for example, preferably introduced into capsules or tablets for oral consumption. Further, the composition can be added directly to the food to enhance the nutrient content.

  In further embodiments, the composition comprises various proteins, vitamins, antibiotics, polysaccharides, and other factors beneficial to the growth of livestock and other ruminants, so that the composition is It may be used as a food supplement.

  In other embodiments of the invention, cells can also be cultured using the compositions of the invention. The cell conditioned medium of the present invention contains factors useful for promoting cell adhesion and cell growth. Further genetic engineering may be used to acclimate cells that may contain higher concentrations of, for example, fibronectin or collagen useful for promoting cell adhesion to a scaffold or culture surface.

  In a further embodiment of the invention, the cell conditioned medium of the invention may be used for pharmaceutical use. The present invention includes a cell culture medium in which growth factors and other proteins are secreted into the medium at a rate that closely resembles that found in vivo by culturing with a three-dimensional tissue construct. The conditioned medium of the present invention is useful for various pharmaceutical applications.

  Finally, the composition of the present invention can also be formulated as a topical formulation for stimulating hair growth.

3.1. Definitions As used herein, the following terms have the following meanings:
Adhesive layer: cells directly attached to a three-dimensional support, or cells indirectly attached through adhesion to a cell directly attached to a three-dimensional support.

  Conditioned medium: A composition containing extracellular proteins and cellular metabolites that has previously supported the growth of the desired eukaryotic cell type. These cells are cultured two-dimensionally or three-dimensionally. Also referred to as “cell conditioned medium” or “cell and tissue conditioned culture medium”.

  Stromal cells: fibroblasts with or without other cells and / or components found in loose connective tissue, including but not limited to endothelial cells, pericytes, macrophages, monocytes, plasma cells, Including mast cells, adipocytes, mesenchymal stem cells, liver reserve cells, neural stem cells, pancreatic stem cells, chondrocytes, cartilage progenitor cells and the like.

  Tissue-specific cells or parenchymal cells: Cells that form the main and discrete tissue of an organ that is distinguished from its supportable framework.

  Three-dimensional framework: a three-dimensional scaffold composed of any material and / or shape, (a) can attach a cell to it (or can be modified to attach a cell to it) (B) A cell that can be grown in two or more layers. A viable three-dimensional stromal tissue is formed by inoculating the support with stromal cells. The framework structure can include a mesh, a sponge, or can be formed from a hydrogel.

  Three-dimensional stromal tissue or viable stromal matrix: A three-dimensional framework that has been inoculated with stromal cells grown on a support. Viable stromal tissue is formed by attaching extracellular matrix proteins produced by stromal cells onto the framework. Viable stromal tissue supports the growth of tissue-specific cells that are subsequently inoculated, thereby forming a three-dimensional cell culture.

  Tissue-specific three-dimensional cell culture or tissue-specific three-dimensional construct: A three-dimensional living stromal tissue that has been inoculated and cultured with tissue-specific cells. In general, tissue-specific cells used to inoculate a three-dimensional stromal matrix are new to mature into “stem” cells (or “replenishment” cells) of the tissue, ie, specific cells that form the parenchyma of the tissue. It is necessary to include cells that produce cells.

The meanings of the following abbreviations are as follows:
BCS: Calf serum
BFU-E: Burst-forming unit-erythroblast
TGF-β: Transforming growth factor-β
DFU-C: Colony forming unit-culture
DFU-GEMM: Colony forming unit-granuloid, erythroblast, monocyte, megakaryocyte
CSF: Colony stimulating factor
DMEM: Dulbecco's Modified Eagle Medium
EDTA: Ethylenediaminetetraacetic acid
FBS: Fetal bovine serum
FGF: Fibroblast growth factor
GAG: Glycosaminoglycan
GM-CSF: Granulocyte / macrophage colony stimulating factor
HBSS: Hanks buffered saline
HS: Horse serum
IGF: Insulin-like growth factor
LTBMC: Long-term bone marrow culture
MEM: Minimum essential medium
PBL: peripheral blood leukocytes
PBS: phosphate buffered saline
PDGF: Platelet-derived growth factor
RPMI 1640: Roswell Park Memorial Institute Medium No. 1640 (GIBCO, Inc., Grand Island, NY)
SEM: Scanning electron microscope
VEGF: Vascular endothelial growth factor

4). Description of drawings
FIG. 2 is a graph depicting glycosaminoglycan and collagen adhesion kinetics over time, with the three-dimensional tissue products Transcyte ™ and Dermagraft®. The adhesion amount of glycosaminoglycan varies depending on the growth time, whereas the adhesion of collagen does not depend on the growth time. Effect of extracellular matrix (recovered from Transcyte ™ and added to human fibroblast and keratinocyte monolayer cultures at dilutions of 1: 2, 1: 5, 1:10 and 1: 100) It is a graph showing. From the figure, the most significant effect was observed with a 1:10 dilution of the matrix. FIG. 5 is a graph showing the relative growth of human fibroblasts and keratinocytes exposed to a conditioned medium (a cell medium that previously supported cell growth with Transcyte ™). An increase in cellular response was evident in a short period of 3 days. Growth in three dimensions compared to basal medium DMEM (containing 2 mM L-glutamine and 1 x antibiotic / antimycotic containing 10% BLS) supplemented with 1 x final concentration of serum-free medium and medium It is a graph showing the effect of 1x conditioned medium (cell culture medium in which cell growth is previously supported by Transcyte (trademark)) on the collagen adhesion layer of the treated cells. Compared to either control, a statistically significant (p = 0.05) increase of almost 50% was recorded in the collagen adhesion layer of cultures treated with conditioned medium for 10 days. It is a graph showing the antioxidant activity in the cell conditioned medium on the human epidermal keratinocytes in culture (the cell medium in which cell growth was previously supported by Transcyte ™). A statistically significant decrease in intracellular oxidation (p <0.0003) of approximately 50% was recorded in human epithelial keratinocytes exposed to conditioned medium that had been pre-supported for 3 days with Transcyte ™.

5). Detailed Description of the Invention The present invention relates to novel compositions comprising any defined or undefined media that have been cultured and conditioned using eukaryotic cell types or three-dimensional tissue constructs, and It relates to a method of using the composition. Cells are cultured in monolayers, beads (ie, two-dimensional) or, preferably, three-dimensionally. The cells are preferably human cells to reduce the risk of an immune response and are stromal cells, parenchymal cells, mesenchymal stem cells, hepatic replacement cells, neural stem cells, pancreatic stem cells, and / or embryonic Contains stem cells. Medium conditioned in cells and in tissue culture contains a variety of naturally secreted proteins such as biologically active growth factors, and those cultured in three dimensions are close to physiological levels Will contain these proteins. The present invention also relates to novel compositions comprising products derived from cell conditioned media, and the use of these compositions.

  The cell “pre-conditioned” medium can be any cell medium that appropriately corresponds to the auxotrophy of the cells to be cultured. Examples of cell culture media include, but are not limited to, the following media: Dulbecco's Modified Eagle Medium (DMEM), Ham F12, RPMI 1640, Iscove Medium, McCoy Medium, and Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture Alan R. Liss, New York (1984) and Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Chiichester, England 1996 (both Other media compositions known to those skilled in the art, including those described in (incorporated herein by reference in their entirety). To the medium can be added any ingredients necessary to support the desired cell or tissue culture. Furthermore, if desired, serum such as bovine serum which is a complex solution of albumin, globulin, growth promoter and growth inhibitor may be added. This serum is pathogen free and must be carefully screened for mycoplasma bacterial, fungal and viral contamination. Serum should generally be obtained from the United States and not from countries where local livestock carries infectious pathogens. It may or may not be desirable to add hormones to the medium.

  Other ingredients such as vitamins, growth and adhesion factors, proteins, etc. can be selected by those skilled in the art according to their specific needs. The present invention can use any type of cells suitable to obtain the desired conditioned medium. Culture may be performed using genetically engineered cells, and the medium may be conditioned. Such cells can be modified to express the desired protein or proteins so that the concentration of the expressed protein in the medium can be optimized for the specific application desired. . In accordance with the present invention, cell and tissue cultures used to conditioned media can be engineered to express target gene products that can confer a great variety of functions. Such functions include, but are not limited to, those that are useful for specific applications such as improved protein expression characteristics similar to physiological responses, wound healing, or suppression of certain proteins such as proteases and lactic acid. Including increased protein expression.

  By manipulating cells, biologically active target gene products, products that exhibit selected biological functions, products that function as reporters of selected physiological conditions, loss of expression of certain gene products Alternatively, it is possible to express a product that improves defects, or a product that provides antiviral, anti-bacterial, anti-microbial, or anti-cancer activity. According to the present invention, the target gene product may be a peptide or protein such as an enzyme, hormone, cytokine, antigen or antibody, a regulatory protein such as a transcription factor or DNA binding protein, a structural protein such as a cell surface protein, or this The target gene product may be a nucleic acid such as a ribosome or an antisense molecule. Target gene products include, but are not limited to, gene products that enhance cell growth. For example, genetic modification can upregulate endogenous proteins, introduce new proteins, or regulate ion concentration by expressing heterogeneous ion channels or altering endogenous ion channel function Can do. By way of example and not limitation, tissue engineered to express gene products (eg, growth factors, hormones, factor VIII, factor IX, neurotransmitters, and enkephalins) delivered systemically Is mentioned.

  In the present invention, the cells are preferably grown on a three-dimensional stromal support and propagated in multiple layers to form a cell matrix. This matrix system approaches the physiological conditions found in vivo to a greater degree than the previously described monolayer tissue culture systems. Dermagraft (registered trademark) (Advanced Tissue Science, Inc., La Jolla, CA) "Dermagraft (registered trademark)" and TransCyte (trademark) (Smith & Nephew, PLC, United Kingdom) "TransCyte (trademark)" Produce growth factors and other proteins that are secreted into the medium at physiological ratios and concentrations. Dermagraft® is composed of allogeneic neonatal fibroblasts cultured on biodegradable polyglactin. TransCyte ™ is a temporary living skin replacement comprising three-dimensional stromal tissue bonded to a transitional covering as described in US Pat. No. 5,460,939. ). Furthermore, three-dimensional tissue cultures that acclimate the cell culture medium are mesenchymal stem cells, liver storage cells, neural stem cells, pancreatic stem cells, and / or embryonic stem cells and / or parenchymal cells that are present in many types of tissues. These tissues may include, but are not limited to, bone marrow, skin, liver, pancreas, kidney, adrenal and neural tissue, and gastrointestinal and genitourinary organs, and circulatory system Including organizations. See U.S. Pat. Nos. 4,721,096, 4,963,489, 5,032,508, 5,266,480, 5,160,490, and 5,559,022. Each of these documents is incorporated herein by reference in its entirety.

5.1. Cell culture media Cell culture compositions are well known in the literature and many are commercially available.

  The components of the preconditioned medium include, but are not limited to, those described below. Furthermore, the concentrations of the components are known to those skilled in the art. See, for example, Methods For Preparation Of Media, Supplements and Substrate for Serum-free Animal Cell Cultures (supra). These components include glutamine, alanine, arginine, asparagine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, triprophane, tyrosine and valine, and derivatives thereof. Amino acids (both D and / or L-amino acids); include acid-soluble subgroups such as thiamine, ascorbic acid, ferric compounds, ferrous compounds, purines, glutathione and monobasic sodium phosphate.

  Further, the above components include sugar, deoxyribose, ribose, nucleoside, water-soluble vitamin, riboflavin, salt, trace metal, lipid, acetate, phosphate, HEPES, phenol red, pyruvate, and buffer.

  Other ingredients commonly used in media compositions include fat-soluble vitamins (including vitamins A, D, E and K), steroids and their derivatives, cholesterol, fatty acids and lipids Tween 80, 2-mercaproethanol pyramidine, Serum (fetus, horse, calf, etc.), protein (insulin, transferrin, growth factor, hormone, etc.), antibiotics (gentamicin, penicillin, streptomycin, amphotericin B, etc.), whole egg ultrafiltrate, and adhesion factor Various supplements such as (fibronectin, vitronectin, collagen, laminin, tenascin, etc.) are included.

  Of course, it may or may not be necessary to add other proteins such as growth factors and adhesion factors to the medium. This is because many cell constructs, particularly the three-dimensional cell and tissue culture constructs described herein, are themselves as described above, as described in more detail in Section 5.8 below. This is because growth and adhesion factors and other products are produced in the medium.

5.2. Cell culture
5.2.1. The cell medium can be conditioned by stromal cells, parenchymal cells, mesenchymal stem cells (lineaged or unrestrained progenitor cells), liver storage cells, neural stem cells, pancreatic stem cells, and / or embryonic stem cells. These cells include, but are not limited to, bone marrow, skin, liver, pancreas, kidney, nerve tissue, adrenal gland, mucosal epithelium, and smooth muscle, to name a few. Fibroblasts and fibroblast-like cells, and other cells and / or components comprising the stroma, may be obtained from either the fetus or the living body, skin, liver, mucous membrane, artery, vein, umbilical cord, As well as from a suitable source such as placenta. Such tissues and / or organs can be obtained by suitable biopsy or at necropsy. In fact, a cadaveric organ allows a rich supply of stromal cells and components.

  Other components, including embryonic stem cells and / or stroma, can be isolated using methods known to those skilled in the art. For example, recently, human embryonic stem cell populations and methods for isolating and using these cells have been described by Keller et al., Nature Med., 5: 151-152 (1999), Smith Curr. Biol. 8: R802-804. (1998); isolated from primordial germ cells, Shamblatt et al., PNAS 95: 13726-1373 (1998), and isolated from undifferentiated germ cells, Thomasson et al., Science 282 : 1145-1147 (1988). Isolation and culture of mesenchymal stem cells is known to those skilled in the art. See Mackay et al., Tissue Eng. 4: 415-428 (1988); William et al., Am Surg. 65: 22-26 (1999). The inoculation of these cells is described in Section 5.3 below. Similarly, neural stem cells can be obtained by the methods described in Flex et al., Nature Biotechnol., 16: 1033-1039 (1998); and Frisen et al., Cell. Mol. Life Sci., 54: 935-945 (1998). Can be isolated.

  Cells are cultured by any method known to those skilled in the art, such as monolayers, beads, or three-dimensional culture, and using any means (ie, culture dish, roller bottle, continuous flow system, etc.). be able to. Cell and tissue culture methods are known to those skilled in the art and are described, for example, in Cell & Tissue Culture: Laboratory Procedures (supra); Freshney (1987), Culture of Animal Cells: A Manual of Basic Techniques (supra). Has been.

  In general, the cell lines used are carefully screened for human and animal pathogens. Depending on the application, such screening may be crucial, in which only cells free of pathogens are tolerated (eg wound healing, food additives, etc.). Pathogen screening methods are known to those skilled in the art. Whether cultivated in two or three dimensions, the cell type will affect the properties of the conditioned medium. A three-dimensional construct is preferred.

5.2.2. Three-dimensional cell culture The stromal cells used in the three-dimensional culture comprise fibroblasts, mesenchymal stem cells, liver storage cells, neural stem cells, pancreatic stem cells, and / or embryonic stem cells. Additional cells and / or components as described in more detail in the specification may or may not be further included.

  Fibroblasts support the growth of many types of cells and tissues in a three-dimensional culture system and are therefore “generic” for culturing any of a wide variety of cells and tissues by inoculating a matrix. An interstitial support matrix can be formed. However, in some cases it may be preferable to use a “specific” stromal support matrix rather than “general”, in which case the stromal cells and components are obtained from a particular tissue, organ or individual. Can do. Furthermore, fibroblasts and other stromal cells and / or components may be derived from the same type of tissue to be cultured in a three-dimensional system. This is because when specific stromal cells are cultured in tissues where specific structural / functional roles can be played, such as arterial smooth muscle cells, neural tissue glial cells, liver Kupffer cells, etc. Would be advantageous.

  When inoculated into a three-dimensional support, stromal cells grow on the framework and attach connective tissue proteins (such as growth factors, regulatory factors and extracellular matrix proteins) that are naturally secreted by the stromal cells. Stromal cells and the connective tissue proteins they naturally secrete substantially encase the framework, thereby forming viable stromal tissue. This stromal tissue supports the growth of tissue-specific cells inoculated in the three-dimensional culture system of the present invention. In fact, when inoculated with tissue-specific cells, the three-dimensional stromal tissue maintains active growth of the culture for an extended period of time. Importantly, the stromal cells continue to grow and divide and become functional because the mesh openings serve as outlets for the stromal cells in culture and the confluent stromal culture does not exhibit contact inhibition. Remains active.

  Growth and regulatory factors are produced in the medium by stromal tissue. Growth factors (including but not limited to αFGF, βFGF, insulin-like growth factor or TGF-beta), or natural or modified blood products or other biologically active biological molecules (including but not limited to (Eg, hyaluronic acid or hormone) enhances colonization or scaffolding of the three-dimensional framework and acclimates the medium.

  Prior to using the culture in vivo, the extent to which stromal cells are grown depends on the type of tissue grown in the three-dimensional tissue culture. Viable stromal tissue conditioned medium can be used as corrective structures by transplanting them in vivo. Alternatively, other types of cells may be inoculated into living stromal tissue and transplanted in vivo. It is also possible to engineer stromal cells to regulate the level of protein product secreted into the medium and to increase the concentration of the product obtained and recovered from the conditioned medium. For example, anti-inflammatory factors such as anti-GM-CSF, anti-TNF, anti-IL-1, and anti-IL-2. In addition, by genetically manipulating stromal cells, the expression of natural gene products that promote inflammation, such as GM-CSF, TNF, IL-1 and IL-2, can be “knocked out” or MHC Expression can also be “knocked out” to reduce the risk of rejection.

  Three-dimensional stromal cell growth will maintain active proliferation of both stromal and tissue-specific cells in culture for a much longer period of time than monolayer systems. In addition, the three-dimensional system supports the maturation, differentiation and secretion of cells in culture in vitro, forms components of adult tissue similar to counterparts present in vivo, and provides physiological ratios. The protein is fixed to the conditioned medium at a ratio closer than before.

Applicants are not obligated to explain the mechanism of action by which many factors inherent to three-dimensional cells and tissues and preparations, ie three-dimensional culture systems, contribute to this success,
(A) The three-dimensional framework provides a larger surface area to which protein adhesion and thus stromal cells adhere;
(B) Because the framework is three-dimensional, stromal cells continue to proliferate actively, as opposed to cells in monolayer culture. In the case of monolayer cultures, when they grow and reach confluence, they exhibit contact inhibition and stop growth and division. Growth of growth and regulatory factors by replicating stromal cells is thought to be partly responsible for stimulating the growth of cultured cells and for regulating differentiation;
(C) the three-dimensional framework allows a partial distribution of cellular components that are more similar to those present in the corresponding tissue in vivo;
(D) By increasing the expected amount of cell growth in a three-dimensional system, a localized microenvironment that helps cell maturation can be established.

(E) the three-dimensional framework maximizes cell-cell interactions by enhancing the motility of migrating cells such as macrophages, monocytes, and possibly lymphocytes in the adhesion layer;
(F) It has been observed that maintaining a differentiated cell phenotype requires not only growth / differentiation factors, but also suitable cell-cell interactions. The present invention provides an excellent conditioned medium by effectively remodeling the tissue microenvironment.

5.3. Establishing a three-dimensional interstitial tissue A three-dimensional support or skeleton can (a) attach a cell to itself (or can be modified to attach a cell to itself); b) Any material and / or shape that allows cells to grow in two or more layers. A variety of materials can be used to form the framework, such materials include, but are not limited to: non-biodegradable materials such as nylon (polyamide), dacron (polyester), Polystyrene, polypropylene, polyacrylate, polyvinyl compounds (eg, polyvinyl chloride), polycarbonate (PVC), polytetrafluoroethylene (PTFE; Teflon), Thermonox (TPX), nitrocellulose, cotton; and biodegradable Materials such as polyglycolic acid (PGA), collagen, collagen sponge, intestinal suture material, cellulose, gelatin, dextran, polyalkanoate and the like. Any of these materials can form a three-dimensional framework, for example, by weaving, braiding, knitting, etc. in a mesh, and this framework can also be any shape desired as an orthodontic structure, such as a tube, It can be formed into a rope or filament. Certain materials such as nylon and polystyrene are unsuitable substrates for cell adhesion. When these materials are used as a three-dimensional framework, it is recommended to enhance the adhesion of the stromal cells to the support by pretreating the framework before inoculation with the stromal cells. For example, before inoculating stromal cells, the nylon framework may be coated with 0.1 M acetic acid and incubated in polylysine, FBS and / or collagen. Polystyrene can be similarly treated with sulfuric acid.

  When trying to transplant a culture conditioned medium in vivo, for example, polyglycolic acid, collagen, collagen sponge, collagen fabric, intestinal suture material, gelatin, polylactic acid, or polyglycolic acid and their copolymers It may be preferred to use a biodegradable matrix. Non-biodegradable materials such as nylon, dacron, polystyrene, polyacrylic ester, polyvinyl, teflon, and cotton are preferred when the culture is maintained for a long period of time or is to be stored frozen. A suitable nylon mesh that can be used in accordance with the present invention is a Nitex, nylon filtration mesh (# 3-210 / 36, Tetko, Inc.) with an average pore size of 210 μm and an average nylon fiber diameter of 90 μm. , NY).

  It contains fibroblasts, mesenchymal stem cells, liver storage cells, neural stem cells, pancreatic stem cells and / or embryonic stem cells, and stromal cells containing or not containing the other cells and components described above Inoculate. Alternatively, cells present in loose connective tissue can be inoculated on a three-dimensional support together with fibroblasts. Such cells include, but are not limited to, smooth muscle cells, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, fat cells and the like. As already explained, fetal fibroblasts can be used to form a “generic” three-dimensional stromal matrix that supports the growth of a wide variety of cells and / or tissues. However, also fibroblasts from a particular individual that are derived from the same type of tissue that is to be cultured and / or that are later grown in media and / or tissue grown in a medium according to the three-dimensional system of the present invention. Can also be prepared by inoculating a three-dimensional framework.

  Thus, in one embodiment of the present invention, specific tissue-specific stromal cells can be cultured. For example, the tertiary for long-term culture of bone marrow in vitro using hematopoietic tissue stromal cells including, but not limited to, fibroblasts, endothelial cells, macrophages / monocytes, adipocytes and reticulum cells The stroma of the original sub-confluent culture can be formed. Hematopoietic stromal cells can be easily obtained from the “buffy coat” formed in the bone marrow suspension by low centrifugal force, eg, centrifugation at 3,000 × g. In the stromal layer constituting the inner wall of the artery, elasticity can be imparted to the protein by further adding a high proportion of non-differentiated smooth muscle cells. Liver stromal cells may include fibroblasts, Kupffer cells, and vascular and biliary endothelial cells. Similarly, the use of glial cells as stromal cells can support the growth of nerve cells and tissues. The glial cells used for this purpose can be obtained by trypsinization or collagenase digestion of the embryo or adult brain (Ponten and Westermark, 1980, Federof, S. Hertz, L., ed. “Advances in Cellular Neurobiology”, Vol. 1, New York, Academic Press, pp. 209-227). Cell proliferation in three-dimensional stromal cell cultures can include proteins (eg, collagen, elastic fibers, reticulum fibers), glycoproteins, glycosaminoglycans (eg, heparan sulfate, chondroitin-4-sulfate, chondroitin-6) -Sulfuric acid, dermatan sulfate, keratin sulfate, etc.), cell matrix, and / or other materials can be further enhanced by adding to the framework or coating the support.

  Furthermore, mesenchymal stem cells (lineaged or unrestricted progenitor cells) are “stromal” cells that are advantageous for inoculation into the framework. The cells differentiate into bone cells, tendon and ligament fibroblasts, bone marrow stem cells, adipocytes, and other cells of connective tissue, chondrocytes, but this differentiation is of course endogenous and supplemented and It depends on regulatory factors and other factors including prostaglandins, interleukins, and natural calones that regulate growth and / or differentiation.

Fibroblasts can be easily isolated by disaggregating suitable organs or tissues that serve as a source of fibroblasts. This is easily accomplished using methods known to those skilled in the art. For example, by mechanically disaggregating tissues or organs and / or treatment with digestive enzymes and / or chelating agents that weaken the binding between adjacent cells, individual cells can be suspended without significant disruption of the cells. The tissue can be dispersed in the suspension. Enzymatic dissociation can be achieved by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes (single or in combination). These digestive enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and / or hyaluronidase, DNase, pronase, dispase and the like. Also, but not limited to, mechanical destruction by a number of methods, including the use of crushers, blenders, sieves, homogenizers, pressure cells, or sonicators, to name a few Can also be achieved. The organization maceration method, for more information, Freshney, Culture of Animal Cells: ... A Manual of Basic Technique, 2 nd Ed, AR Liss, Inc., New York, 1987, Ch 9, pp see 107-126 .

Once the tissue is in a suspension of individual cells, the suspension can be fractionated into small populations from which fibroblasts and / or other stromal cells and / or components can be obtained. . This also includes, but is not limited to, cloning and selection of certain types of cells, selective destruction of unwanted cells (negative folds), separation based on cell aggregation differences in mixed populations, freeze-thaw methods, mixed populations Standard cells including cell adhesion differences, filtration, conventional and differential centrifugation, centrifugal sieving (countercurrent centrifugation), unit gravity separation, countercurrent distribution, electrophoresis, and fluorescence activated cell sorting It can also be achieved using separation methods. For more information on clonal selection and cell separation methods, Freshney, Culture of Animal Cells: see ^{A Manual of Basic Technique, 2 nd} Ed, AR Liss, Inc., the New York, 1987, Ch 11, pp 137-168... I want to be.

Fibroblast isolation can be performed, for example, as follows: Serum is removed by carefully washing fresh tissue in Hanks buffered saline (HBSS) and minced. The minced tissue is incubated for 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After incubation, dissociated cells are suspended, pelleted by centrifugation, and applied to culture dishes. Since all fibroblasts adhere before other cells adhere, appropriate stromal cells can be selectively isolated and grown. The isolated fibroblasts are then grown to confluence, harvested from the confluent culture, and inoculated into a three-dimensional matrix (Naughton et al., 1987, J. Med. 18 (3 and 4) 219 -250). For example, inoculation of a three-dimensional framework of stromal cells at a high concentration of about 10 ^{6 to} 5 × 10 ⁷ cells / ml establishes a three-dimensional stromal tissue in a shorter period of time.

  After stromal cell inoculation, the three-dimensional framework must be incubated in a suitable nutrient medium. As already mentioned, many commercially available media are suitable for use, such as RPMI 1640, Fisher's medium, Iscove's medium, MacCoy's medium. It is important to maximize proliferative activity by suspending or suspending the three-dimensional stromal cell culture in the medium during the incubation period. The culture is periodically “feeded” and the conditioned medium of the present invention is collected and then processed as described in Sections 5.6 and 5.7 below. Thus, depending on the tissue to be cultured and the type of collagen desired, suitable stromal cells can be selected and inoculated into the three-dimensional matrix.

  During incubation of the three-dimensional stromal cell culture, proliferating cells can be released from the matrix. These released cells adhere to the walls of the culture vessel where they can continue to grow and form a confluent monolayer. This must be prevented, for example, by removing the released cells during feeding or by transferring the three-dimensional stromal culture to a new culture vessel. The presence of a confluent monolayer in the container will lead to a “stop” of cell growth in the three-dimensional matrix and / or culture. Removal of confluent monolayers or transfer to fresh media in a new container preserves the growth activity of the three-dimensional culture system. It should be noted that if necessary, the conditioned medium of the present invention can be treated so that it does not contain any whole cells (except of course when whole cells are used for a specific application). Such removal or transplantation must be performed in all culture vessels with a stromal monolayer exceeding 25% confluence. Other than this, stirring the culture system will prevent the released cells from adhering, or instead of feeding the culture regularly, so that fresh medium will flow continuously through the culture system. It is also possible to prepare a culture system. Adjusting the flow rate to maximize growth in the three-dimensional culture, while at the same time washing and removing the cells released from the culture, these cells attach to the vessel wall and grow to confluence Do not.

  Other cells, such as parenchymal cells, can also be seeded and grown in three-dimensional living stromal tissue.

5.4. Inoculation of tissue-specific cells into a three-dimensional stromal matrix and maintenance of the culture Once the three-dimensional stromal cell culture has reached a suitable degree of growth, the tissue-specific cells to be cultured (parenchyma) Cells) or additional cells such as surface layer cells can also be seeded into living stromal tissue. By growing cells on viable stromal tissue in vitro, it forms a culture counterpart of natural tissue and acclimates the medium by producing extracellular products in the medium at a ratio similar to physiological levels. To do. A high concentration of inoculum is advantageous, which increases the growth of the culture much faster than that of a low concentration. The cells selected for inoculation depend on the tissue to be cultured and are not limited, but include, but are not limited to, bone marrow, skin, liver, pancreas, kidney, nerve tissue, adrenal gland, mucosal epithelium, Includes smooth muscle. Such cells produce characteristic extracellular proteins, such as predetermined growth factors, in the medium, thereby obtaining a medium optimized for a specific tissue for a particular application.

  For example, but not limited to, various epithelial cells can be cultured on three-dimensional viable stromal tissue. Examples of such epithelial cells include, but are not limited to, keratinocytes, oral mucosa and gastrointestinal (G.I.) duct cells. Such epithelial cells are isolated by enzymatic treatment of the tissue according to methods known to those skilled in the art, and then these cells are cultured and expanded so that the epithelial cells are three-dimensional stromal support cells. Apply to matrix. The presence of a stromal support provides growth factors and other proteins that promote normal division and differentiation of epithelial cells.

  In general, the inoculum contains cells that produce “stem” cells (also called “replenishment” cells) of the tissue, ie, new cells that mature into specific cells that form various components of the tissue. There must be.

The parenchyma or other surface layer cells used for inoculation are prepared by disaggregating the desired tissue using the standard method of stromal cell acquisition described in Section 5.3 above. Can be obtained from The whole cell suspension itself can also be used to inoculate three-dimensional live stromal tissue. As a result, regenerative cells contained in the homogenate grow, mature and differentiate properly on the matrix, while non-regenerative cells do not grow, mature and differentiate. Alternatively, certain types of cells can be isolated from a suitable fraction of the cell suspension using standard stromal cell fractionation methods described in Section 5.1. If “stem” cells or “supplemented” cells can be easily isolated, they can be used to inoculate preferably a three-dimensional stromal support. For example, when culturing bone marrow, the three-dimensional stroma can be inoculated with either fresh bone marrow cells or bone marrow cells of a cryopreserved sample. When culturing skin, the three-dimensional stroma can be inoculated with melanocytes and keratinocytes. In addition, when culturing the liver, hepatocytes can be inoculated into the three-dimensional stroma. When the pancreas is cultured, pancreatic endocrine cells can be inoculated into the three-dimensional stroma. How that can be used to get parenchymal cells from various tissues, specifically, Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2 nd Ed., AR Liss, Inc., New York, 1987, Ch. 20 pp. 257-288.

  In fact, the various ratios of various collagens attached to the stromal matrix prior to inoculation can affect the growth of tissue-specific cells that are subsequently inoculated. For example, for optimal growth of hematopoietic cells, the matrix preferably comprises type III collagen, type IV collagen and type I collagen in a ratio of about 6: 3: 1 in the initial matrix. In a three-dimensional skin culture system, it is preferred to attach type I and type III collagen to the initial matrix. The ratio of collagen types to be attached can be manipulated or enhanced by selecting fibroblasts that produce suitable collagen types. This can be achieved using a monoclonal antibody of the appropriate isotype or subclass that can activate complement and identify a particular collagen type. These antibodies and complement can be used to perform negative selection of fibroblasts that express the desired collagen type. Alternatively, the stromal cells used to inoculate the matrix may be a mixture of cells that synthesize the desired appropriate collagen type. The distribution and origin of each collagen type is shown in Table 1.

  During the incubation, the three-dimensional cell culture system should be suspended or suspended in a nutrient medium. The culture must be replenished regularly with fresh medium. Again, care should be taken that the cells released from the culture do not adhere to the walls of the container, where they can proliferate to form a confluent monolayer. It is believed that cell release from a three-dimensional culture occurs more easily when diffusing tissue is cultured as opposed to structural tissue. For example, the three-dimensional skin culture of the present invention is histologically and morphologically normal, and the apparent dermal and epithelial layers do not release cells into the surrounding medium. In contrast, the three-dimensional bone marrow culture of the present invention releases mature, non-adherent cells into the medium in a manner similar to that such cells are released in the bone marrow in vivo. As explained above, the released cells should attach to the culture vessel and form a confluent monolayer, and the growth of the three-dimensional culture will “stop”. This can be done by feeding the nutrients or removing the free cells when transferred to a new container, stirring the culture to prevent the free cells from sticking to the container walls, or into the culture. This can be avoided by continuously flowing fresh media at a rate sufficient to supply nutrients and remove free cells. As stated above, the conditioned medium is treated to avoid the inclusion of whole cells and cell debris if necessary.

  Growth and activity of cells in culture is affected by various growth factors such as insulin, growth hormone, somatomedin, colony-stimulating factor, erythropoietin, epidermal growth factor, hepatic erythropoietin-like factor (hepatopoietin) and hepatocyte growth factor May receive. Other factors that regulate growth and / or differentiation include prostaglandins, interleukins and natural silones.

5.5. In another embodiment of a genetically engineered construct, a three-dimensional construct that conditioned the medium acts as a vehicle for introducing into the medium, for example, a gene product that promotes repair and / or regeneration of tissue defects. sell. The cells can be genetically engineered to express inflammatory mediators such as IL-6, IL-8 and G-CSF. Alternatively, the cells may be genetically engineered to express anti-inflammatory factors such as anti-GM-CSF, anti-TNF, anti-IL-1 and anti-IL-2.

  In another embodiment, the cells may produce TGF-β to stimulate cartilage production, other factors such as BMP-13 to promote cartilage formation, or stromal cell migration and / or For example, in the production of stimulating factors that promote matrix adhesion, genetic manipulation can be performed so that a gene exhibiting a therapeutic effect is expressed in the medium. Since this construct contains eukaryotic cells, the gene product is appropriately expressed and processed to produce an active product. Preferably, the expression control element used can regulate the expression of the gene so that the product can be oversynthesized in culture. Generally selected transcription promoters, particularly promoter elements, depend in part on the tissue and cell type being cultured. Cells and tissues capable of secreting proteins are preferred (eg, rich in rough endoplasmic reticulum and Golgi complex organelles). The overproduced gene product is then secreted into the conditioned medium by the genetically engineered cells.

  The cells used to condition the medium can be engineered to regulate one or more genes. Alternatively, the regulation of gene expression may be transient or long-term, and the activity of the gene may be non-inducible or inducible.

  Cells conditioned to media can also be genetically engineered to “knock out” the expression of factors that promote inflammation. A negative regulation method for reducing the expression level of the target gene or the activity level of the target gene product will be described later. As used herein, “negative regulation” means that the level and / or activity of the target gene product is reduced when compared to the level and / or activity of the target gene product without the regulatory treatment. Point to. Expression of a gene that is negative for the cell can be reduced or knocked out using a variety of techniques, eg, completely inactivating the gene using standard homologous recombination techniques (generally “ The expression can be blocked by “knockout”. Usually, an exon that encodes a critical region of a protein (or an exon 5 ′ to that region) is cleaved by a positive selectable marker (eg, neo) to prevent normal mRNA production from the target gene. Inactivation of the gene occurs. A gene can also be inactivated by making an insertion to delete or inactivate part of the gene or by deleting the entire gene. By using a construct with two regions that are homologous to the target gene and that are quite distant in the genome, the sequence flanked by the two regions can be deleted (Mombaerts et al. 1991, Proc. Nat. Acad. Sci. USA 88: 3084-3087). Alternatively, the gene may be inactivated by deleting upstream or downstream expression elements.

In addition, antisense molecules and ribozyme molecules that suppress the expression of the target gene can be used in the present invention to reduce the activity level of the target gene. For example, antisense RNA molecules that suppress the expression of major histocompatibility gene complexes (HLA) have been shown to act most diversely with respect to immune responses. In addition, suitable ribozyme molecules are described, for example, in Haseloff et al., 1988, Nature 334 : 585-591; Zaung et al., 1984, Science 224 : 574-578; and Zaug and Cech, 1986, Science 231 : 470-475. Can be designed. Furthermore, triple helix molecules can be utilized to reduce the activity level of the target gene. These techniques are described in detail by LGDavis et al. (Eds.), Basic Methods in Molecular Biology, 2nd edition, Appleton & Lange, Norwalk, Conn. 1994.

  Methods that may be useful for genetically engineering cells of the present invention are known in the art and are described in further detail in Applicants' U.S. Pat. Nos. 4,963,489 and 5,785,964, these patents. The disclosure of which is incorporated herein by reference. For example, a recombinant DNA construct or vector containing an exogenous nucleic acid (eg, one encoding a gene product of interest) can be constructed and used to transform or transfect stromal cells of the invention. . Such transformed or transfected cells that carry the exogenous nucleic acid and are capable of expressing the nucleic acid are selected and expanded as clones within the three-dimensional construct of the present invention.

  Methods for preparing a DNA construct containing the gene of interest, methods for transforming or transfecting cells, and methods for selecting cells that carry and express the gene of interest are known in the art. For example, Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Willey Interscience, NY; and Sambrook et al., See the technique described in 1989, Molecular Cloning: A Laboraotry Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  The cells can be manipulated using any of a variety of vectors, such as integrative viral vectors such as retroviral vectors or adeno-associated viral vectors; or papillomavirus vectors, SV40 vectors, adenovirus vectors. Non-integrating replicative vectors such as, but not limited to, replication deficient viral vectors. If transient expression is desired, non-integrating vectors and replication-deficient vectors may be preferred. This is because in these systems, either inducible or constitutive promoters can be used to regulate the expression of the gene of interest. Alternatively, transient expression can be achieved using an integrative vector provided that the gene of interest is regulated by an inducible promoter. Other methods for introducing DNA into cells include the use of liposomes, lipofection, electroporation, particle gun, or direct DNA injection.

  The cells are preferably transformed or transfected with a nucleic acid such as DNA, and in particular by one or more suitable expression control elements such as promoter or enhancer sequences, transcription terminators, polyadenylation sites, and selectable markers (ie , In a functionally connected manner). After introducing the foreign DNA, the genetically engineered cells can be grown in an enriched medium and then switched to a selective medium. A selectable marker in the foreign DNA confers resistance to selection, allowing the cell to stably integrate, for example, foreign DNA on a plasmid into its chromosome and proliferate to form a focus. The focus can then be cloned and expanded into cell lines. This method can advantageously be used to create cell lines that express the gene product in the culture medium.

  Any promoter can be used to drive the expression of the inserted gene. For example, viral promoters include, but are not limited to, CMV promoter / enhancer, SV40, papilloma virus, Epstein-Barr virus, elastin gene promoter and β-globin. Preferably, the regulatory element used to regulate the expression of the gene of interest is one that allows for regulation of the expression of the gene so that the product is synthesized only when needed in vivo. Where transient expression is desired, it is preferred to use a constitutive promoter in non-integrating and / or replication deficient vectors. Alternatively, inducible promoters can be used to drive insertional gene expression as needed. Inducible promoters can also be incorporated into integrative and / or replicative vectors. For example, inducible promoters include, but are not limited to metallothionein and heat shock proteins.

  According to one embodiment, the inducible promoter used to express a foreign gene of interest is a negative promoter of a regulatory protein disclosed herein, which can be stored by cryopreservation and subsequent thawing. Be guided. For example, TGF-β, VEGF, or various known heat shock protein promoters can be used as expression control elements, ie, they express the desired gene product in a tissue construct that acclimates the cell culture medium. Therefore, it can be linked in a functional manner to the foreign gene of interest.

Various methods can be used to achieve homeostatic or transient expression of the generated gene product in the cell. For example, the transkaryotic transplantation method described by Seldon et al., 1987, Science 236 : 714-718 can be used. As used herein, “transkaryotic” indicates that the nucleus of the transplanted cell has been altered by the addition of a DNA sequence by stable or transient transfection. Preferably, the cells are adapted to transform such gene products transiently and / or under inducible regulation during the postoperative recovery phase, or as chimeric fusion proteins immobilized to stromal cells (eg extracellular It is made to be expressed as a chimeric molecule composed of intracellular and / or transmembrane domains of a receptor or receptor-like molecule fused to a gene product as a domain.

  In addition, it may be desirable to prepare constructs that include an extracellular matrix that includes exogenous gene products, growth factors, regulatory factors, and the like that are subsequently present in the conditioned medium. This embodiment synthesizes human extracellular matrix as human stromal cells grow on a three-dimensional support framework in the same manner as it is produced in normal human tissue. Based on the knowledge of adhering to This extracellular matrix is not only secreted locally by the cells and binds cells and tissues together, but also affects the development and behavior of the cells it contacts. The extracellular matrix contains a variety of connective tissue proteins, such as fiber-forming proteins that are intertwined with a hydrated gel of glycosaminoglycan chains that make up the network. Glycosaminoglycans are long, negatively charged, heterogeneous classes of polysaccharide chains that (except for hyaluronic acid) are covalently linked to proteins to form proteoglycan molecules. According to this embodiment of the invention, the stromal cells can be genetically engineered to express the desired gene product or a modified version of the gene product, which gene product or modified version thereof is in the extracellular matrix. And will eventually be present in the cell culture medium.

5.6. The harvested cells of the conditioned medium may be cultured by any means known in the art. Preferably, the cells are cultured in an environment that allows aseptic processing and handling. Conventional means of cell and tissue culture are limited by the need for human media management and processing. This limits the amount of cells and tissues that can be cultured at one time, and thus the volume of conditioned cell medium that can be obtained at one time. For this reason, large scale growth (mass leveling) can be achieved using a device for aseptic large-scale culture such as that described in commonly assigned US Pat. No. 5,763,267 (“the '267 patent”). It is preferred that the medium is conditioned in a manner capable of providing a medium. U.S. Pat. No. 5,763,267 is incorporated herein by reference in its entirety for all purposes. Using the sterile closed system described in the '267 patent (U.S. Pat.No. 5,763,267), the pre-conditioned medium moves from the fluid reservoir to the inlet manifold and enters the continuous flow system. And is evenly distributed in the culture and is useful for culturing three-dimensional cultures of cells and tissues such as fluid systems such as Dermagraft®. In particular, the device described in the '267 patent includes a plurality of flexible or semi-flexible processing chambers including one or more individual culture pockets, a plurality of rigid spacers, a fluid inlet manifold, a fluid outlet manifold ( an outlet inlet manifold), a fluid reservoir, and means for transporting fluid within the system.

  During processing, the liquid medium moves from the fluid reservoir to the inlet manifold, and then distributes the medium evenly to each of the connected processing chambers and in-culture pockets. A fluid outlet manifold is also provided to ensure that bubbles generated during processing are removed from the processing chambers to ensure that each processing chamber is filled reliably and evenly. The processing chamber is flexible or semi-flexible so that the end user can easily handle the washed and applied cultured implants. Due to the flexibility of the processing chamber, a rigid spacer is also provided, which ensures that the fluid is evenly distributed within the chamber during processing. At the appropriate time (ie, once the medium is conditioned until extracellular proteins such as growth factors reach the desired level in the medium), the “conditioned” medium is pumped from the system, processed and used. Preferably, the conditioned cell medium is recovered from the device at a later stage of tissue growth and when the secretion levels of certain growth factors and connective tissue proteins are at a maximum level (see FIG. 1). In one preferred embodiment, media conditioned with three-dimensional cell culture is harvested on days 10-14 of culture after exposing the media to the cells.

  In another embodiment, the three-dimensional tissue is a three-dimensional tissue as described in US Pat. No. 5,843,766 (“the '766 patent”), which is hereby incorporated by reference in its entirety for all purposes. Incubate in an apparatus for aseptic growth of the culture. The '766 patent discloses a tissue culture chamber that is a container for the growth of three-dimensional tissue that is grown in the same sterile container and stored in frozen form. Can be transported to end users. The tissue culture chamber includes a container having a substrate therein designed to facilitate three-dimensional tissue growth on the surface thereof. The container includes an inlet and an outlet that assist in inflow and outflow of the culture medium. The container also includes at least one fluid distributor. In one embodiment, the fluid distributor is a baffle, which is used to distribute the medium flow within the chamber to create one continuous, uniform three-dimensional tissue piece. In the second embodiment, the fluid distributor is a combination of a deflector plate, a distribution channel, and a fluid channel. In each embodiment, the container further comprises a seal to ensure that the interior of the chamber is a sterile environment during tissue growth and storage. Again, the medium is preferably recovered from the device at a later stage of tissue growth and when the secretion levels of growth factors and connective tissue proteins are at their maximum levels (see FIG. 1). In one preferred embodiment, the medium conditioned by the three-dimensional cell culture is harvested 10-14 days in culture after exposing the medium to the cells.

5.7. Conditioned Medium Concentration After collecting the conditioned medium, it may be necessary to further process the resulting supernatant. Such treatment includes defiltration using a water flux filtration device or using the method described in Cell & Tissue Culture: Laboratory Procedures, supra, pp 29D: 0.1-29D: 0.4. concentration by defiltration), but is not limited thereto.

  Further, the medium may be concentrated 10 to 20 times using a pressure concentrator (Amicon, Beverly, MA) equipped with a 10,000 ml cut-off filter.

  The conditioned medium may also be further processed for product isolation and purification, such as removing unwanted proteases. The methods used to isolate and purify the product so that optimal biological activity is maintained will be readily apparent to those skilled in the art. For example, it may be desirable to purify growth factors, regulatory factors, peptide hormones, antibodies, and the like. Such methods include gel chromatography of conditioned media [using a carrier such as Sephadex], metal chelate affinity chromatography using an insoluble carrier such as ion exchange, cross-linked agarose, HPLC purification and hydrophobicity. Examples include, but are not limited to, interaction chromatography. Such techniques are described in great detail in Cell & Tissue Culture: Laboratry Procedures, supra. Of course, depending on the intended use of the conditioned medium and / or the product derived therefrom, appropriate measures must be taken to maintain sterility. Alternatively, sterilization may be necessary, which can be accomplished by methods known to those skilled in the art, such as heating and / or filter sterilization, taking care to maintain the desired biological activity.

5.7.1. Isolation of collagen As mentioned above, the conditioned medium of the present invention contains a number of products, which can be isolated and purified from the conditioned medium. For example, human dermal fibroblasts synthesize and secrete collagen precursors, and fractions of these precursors are taken up by a three-dimensional extracellular matrix. This uptake requires removal of terminal peptides (N- and C-peptides), which significantly reduces the solubility of the collagen molecule (the remainder of the secreted collagen has no proteolytic degradation) To remain in solution). In general, soluble collagen can be obtained at high salt concentrations under neutral pH conditions. Kielty, CM, I. Hopkinson et al. (1993), Collagen: The Collagen Family: Structure, Assembly, and Organization in the Extracellular Matrix, Connective Tissue and Its Heritable Disorders: molecular, genetic and medical aspects.PMRoyce and B. Steinmann. New York, Wiley-Liss, Inc .: See 103-149. Applicants have prepared three-dimensional tissue by measuring the amount of collagen secreted into the extracellular matrix of tissue cultured in the presence of serum-free medium, medium or three-dimensional conditioned medium, and Data are presented that show the effect of conditioned medium (medium that previously supported the growth of three-dimensional cultured cells) in composition (see Section 6.3). The conditioned medium of the present invention significantly increases tissue collagen accumulation in vitro, as shown in FIG.

  In addition, Applicants have surprisingly found that collagen is secreted during the culturing process, not present uniformly, but with gradually increasing levels (see FIG. 1). . Therefore, the applicants applied this knowledge in the recovery of collagen.

  It should be understood that the following protocols are presented for purposes of illustration and can be modified using methods known to those skilled in the art. To purify the collagen, 240 mL of medium conditioned with fibroblasts is added to 240 mL of 5 M NaCl (1: 1 ratio of medium to salt) and allowed to settle for 16 hours at 4 ° C. Centrifuge the supernatant at 4000 xg for 20 minutes. Discard the supernatant. The pellet is washed with 10 mL of a solution of 50 mM Tris-HCl (pH 7.5) and 2.4 M NaCl. Centrifuge at 4000 xg for about 20 minutes. Discard the supernatant. Resuspend the pellet in 10 mL of 0.5 M acetic acid. To remove the propeptide, add 0.1 mL pepsin (100 mg / mL) (Sigma Chemical, St. Louis, MO) and digest for 16 hours at 4 ° C. (this removes the propeptide, The triple helix remains intact). Centrifuge the supernatant at 4000 xg for 20 minutes. Collect the supernatant and discard the pellet. Add 2.1 mL of 5 M NaCl and 0.5 M acetic acid to a final volume of 15 mL (final NaCl concentration is 0.7 M). Precipitate at 4 ° C. for about 16 hours. Centrifuge the supernatant at 4000 xg for 20 minutes and discard the supernatant. Dissolve the pellet in 0.5 mL of 0.5 M acetic acid solution. The purity of the collagen should be at least 90% and can be analyzed by standard methods known in the art such as SDS-PAGE.

5.8. Application using conditioned medium
5.8.1. Application to Wound Healing The conditioned medium of the present invention can be treated to promote wound and burn healing. When tissue is damaged, peptidic growth factors (which exhibit many biological activities) are released into the wound to promote healing. Wound healing is a complex process involving several stages that seals the tears in a controlled manner and forms a tissue that is functionally sufficient as an envelope. This process begins with hemostasis followed by an inflammatory phase involving neutrophils and macrophages. This process continues with the development and development of granulation tissue and re-epithelialization, closing the wound. Subsequently, scar tissue is formed and reconstructed to approximately the original anatomy over the following months. Ideally, scar tissue should be as small as possible so that healthy tissue can be formed, that is, a tissue having a functionally sufficient capacity that is histologically and physiologically similar to the original normal tissue.

  Each stage of the healing process is regulated by intracellular interactions and regulatory mechanisms such as cytokines, growth factors, and inflammatory mediators, as well as intracellular contact mechanisms. For example, inflammatory mediators such as IL-6, IL-8 and G-CSF induce lymphocyte differentiation and acute phase proteins, as well as neutrophil infiltration, maturation and activation, which are It is an important process in the inflammatory stage. Other examples of regulatory proteins involved in the process of wound healing are VEGF (inducing angiogenesis and granulation tissue formation during inflammation), BMP (inducing bone formation), KGF (activating keratinocytes), And TGF-β1, which induces extracellular matrix accumulation. Table 2 (below) shows in the conditioned medium of the present invention that previously supported the growth of cells grown in Dermagraft® tissue culture as determined by ELISA (enzyme linked immunoassay). The concentrations of several growth factors are shown. The table below is not a list containing all of the factors, but is presented only to further characterize the conditioned media by presenting some concentrations of biologically active factors present in the media of the invention. Should be understood.

  In chronic wounds, the healing process is interrupted at some point after hemostasis and before re-epithelialization, and re-initiation seems impossible. Although most of the inflammation seen in the wound bed is associated with infection, this inflammation results in a protease-rich environment that degrades regulatory proteins and thus interferes with the wound healing process.

  Various methods have been used to quantify and characterize the major molecular components secreted by fibroblasts found in three-dimensional tissue cultures, TransCyte ™ and Dermagraft ™. Human matrix proteins and glycosaminoglycans (GAGs) present in TransCyte ™ and Dermagraft® include type I collagen, type III collagen, fibronectin, tenascin, decorin, versican, beta glycan, syndecan, and Other ingredients may be mentioned but are not limited to them (data not shown). These secreted proteins and GAGs not only perform major structural functions, but also stimulate cell division, migration, adhesion and signal transduction. Fig. 1 shows glycosaminoglycan accumulation (accumulation capacity is dependent on growth period) and collagen accumulation (accumulation capacity is not dependent on growth period) in a three-dimensional growth system. These components were measured by ELISA, Western blot analysis, immunohistology and PCR. For example, some components found in TransCyte ™ include type I collagen, type III collagen and type VII collagen (RNA), fibronectin, tenascin, thrombospondin 2, elastin, proteoglycan, decorin, versican, and others (Data not shown). The activity of these components in tissue development, healing and normal function has been described in detail. In addition, Applicants describe the specific effects of biologically produced human matrices on cell function in vitro. For example, Applicants note that cell proliferation is increased by the addition of a biologically produced matrix. To examine its effect on cell proliferation, the matrix was physically removed from TransCyte ™ and Dermagraft ™ and added to human fibroblast and keratinocyte monolayer cultures at various dilutions . The results of increased cell proliferation are shown in FIG.

  Furthermore, 6.3. As described in detail in the section, Applicants have identified the effect of three-dimensional culture conditioned medium on the preparation and composition of three-dimensional tissue in the presence of serum-free medium, medium or three-dimensional culture conditioned medium. It should be noted that the amount of collagen secreted into the extracellular matrix of the tissue cultured in (1) was measured. The effect of three-dimensional culture conditioned medium on the preparation and composition of three-dimensional tissue measures the amount of collagen secreted into the extracellular matrix of tissues cultured in serum-free medium, medium or three-dimensional conditioned medium It was investigated by. As shown in FIG. 4, the conditioned medium of the present invention significantly increases tissue collagen accumulation in vitro. The present invention includes many of the regulatory proteins that are considered important for wound healing and have been shown to be deficient in in vivo models of wound healing. Furthermore, in some medical conditions such as diabetes, some supplies of regulatory proteins necessary for wound healing are inadequate. For example, in mouse models of non-insulin dependent diabetes (eg, db / db mice), VEGF and PDGF secretion and PDGF receptor expression in wounds are all reduced compared to levels in normal mouse wounds I know that.

  Also, since many such growth factors have been found in Applicants' conditioned cell media, the conditioned media provided by the present invention can be of other types, such as traumatic or congenital. It is also useful for tissue damage. In this case, repair and / or regeneration of tissue defects or damage is desired. Such growth factors include, for example, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bone morphogenetic protein (BMP), transforming growth factor (TGF), and blood vessels Those that modulate formation include vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic FGF, as well as angiogenic and anti-angiogenic factors. Stress proteins such as GR78 and MSP90 induce growth factors such as TGF-β. TGF-β (including TGFβ-1, TGFβ-2, TGFβ-3, TGFβ-4 and TGFβ-5) regulates proliferation and differentiation and promotes wound healing (Noda et al., 1989, Endocrin. 124). : 2991-2995; Goey et al., 1989, J. Immunol. 143: 877-880; Mutoe et al., 1987, Science 237: 1333-1335). Mitogens such as PDGF increase the rate of cytoplasm and granulation in tissue formation (Kohler et al., 1974, Exp. Cell. Res. 87: 297-301). As stated earlier, the cells are preferably of human origin in order to minimize immunogenicity problems.

  Since the conditioned medium of the present invention contains a number of such wound healing factors, the conditioned medium can be used for skin wounds, fractured bones, gastric ulcers, pancreas, liver, kidneys, spleen, vascular damage, And advantageously used in the treatment of wounds and burns, including other internal wounds. Furthermore, this conditioned medium can be combined with other pharmaceutical ingredients such as antibiotics and analgesics. Embodiments include formulating a conditioned medium with salves and ointments for topical application. In fact, the conditioned medium of the present invention has been shown to induce proliferation of human fibroblasts and keratinocytes. Increased cellular response was confirmed by cells exposed to conditioned media for at least 3 days in vitro (FIG. 3).

  Alternatively, the conditioned medium may be combined with a bandage (adhesive or non-adhesive) to enhance and / or promote wound healing. The conditioned medium can be used in any state, ie liquid or solid, lyophilized or dried into a powder, used as a film for topical wound healing and anti-adhesion applications, as an injection (See PCT WO96 / 39101; which is incorporated herein by reference in its entirety).

  Alternatively, the conditioned medium of the present invention is described in US Pat. Nos. 5,709,854, 5,516,532, 5,654,381 and WO98 / 52543, each of which is incorporated herein by reference in its entirety. It may be formulated with a polymerizable or crosslinkable hydrogel. Examples of materials that can be used to form a hydrogel include modified alginate. Alginate is a carbohydrate polymer isolated from seaweed and exposed to divalent cations such as calcium, as described, for example, in WO94 / 25080 (the disclosure of which is incorporated herein by reference). By doing so, it can be crosslinked to form a hydrogel. Alginate is ionically crosslinked in water in the presence of divalent cations at room temperature to form a hydrogel matrix. As used herein, the term “modified alginate” refers to alginate that has been chemically modified and has altered hydrogel properties.

  In addition, polysaccharides that gel upon exposure to monovalent cations (including bacterial polysaccharides such as gellan gum and vegetable polysaccharides such as carrageenan) are similar to the methods available for cross-linking alginate described above. To form a hydrogel.

  Modified hyaluronic acid derivatives are particularly useful. As used herein, the term “hyaluronic acid” refers to naturally and chemically modified hyaluronic acid. Modified hyaluronic acid can be designed and synthesized by routine chemical modifications to adjust the rate and extent of crosslinking and biodegradability.

  Also useful are hydrogel precursors crosslinked by covalent bonds. For example, a water-soluble polyamine such as chitosan can be crosslinked with a water-soluble diisocyanate such as polyethylene glycol diisocyanate.

  Alternatively, a polymer containing a substituent that crosslinks by a radical reaction after contacting with a radical initiator may be used. For example, polymers containing photochemically crosslinkable ethylenically unsaturated groups are available, such as disclosed in WO93 / 17669, the disclosure of which is incorporated herein by reference. In this embodiment, a water soluble macromer is provided that includes at least one water soluble region, a biodegradable region, and at least two free radical polymerization regions. An example of these macromers is PEG-oligolactyl-acrylate, where the acrylate group is polymerized using a radical initiating system such as an eosin dye or by brief exposure to ultraviolet or visible light. Furthermore, water-soluble polymers that induce photochemically polymerizable cinnamoyl groups, such as those disclosed in Matsuda et al., ASAID Trans., 38: 154-157 (1992), can be used.

  Preferred polymerizable groups are acrylates, diacrylates, oligoacrylates, dimethacrylates, oligomethacrylates and other biologically acceptable photopolymerizable groups. Acrylate is the most preferred active species polymerizable group.

  Natural and synthetic polymers can be modified using chemical reactions available in the art, such as those described in March, “Advanced Organic Chemistry”, 4th edition, 1992, Wiley-Intersicence Publication, New York.

  The polymerization is preferably initiated using a photoinitiator. Useful photoinitiators are those that can be used to initiate macromer polymerization in a short time, at least within minutes, and most preferably within seconds, without causing cytotoxicity.

  A number of dyes can be used for photopolymerization. Suitable dyes are known to those skilled in the art. Preferred dyes include erythrosine, phloxime, rose bengal, thonine, camphorquinone, ethyl eosin, eosin, methylene blue, riboflavin, 2,2-dimethyl-2-phenylacetophenone, 2-methoxy-2-phenyl Acetophenone, 2,2-dimethoxy-2-phenylacetophenone, other acetophenone derivatives, and camphorquinone. Suitable cocatalysts include amines such as N-methyldiethanolamine, N, N-dimethylbenzylamine, triethanolamine, tritylamine, dibenzylamine, N-benzylethanolamine, -isopropylbenzylamine. Triethanolamine is a preferred cocatalyst.

  In another embodiment, the conditioned medium of the present invention, or certain extracellular matrix proteins that occur in the medium, can be used to provide an excellent material for coating sutures. Naturally secreted extracellular matrix includes type I and type III collagen, fibronectin, terascin, glycosaminoglycan, acidic and basic FGF, TGF-α and TGF-β, KGF, versican, decorin and various A conditioned medium containing other secreted human skin matrix proteins is provided. Similarly, the cell conditioned media of the present invention and the extracellular matrix proteins derived from the conditioned media coat conventional implant devices, including vascular prostheses, in a surgical approach to correcting bodily defects. Can be used to create a superior implant device. The implant should be made from a biocompatible, inert material that replaces or substitutes for the missing function, and should be made from either a non-biodegradable material or a biodegradable material. By coating the transplantation device with a medium containing these extracellular proteins, the graft will have a suitable cell adhesion, resulting in better tissue at the transplant site. Thus, sutures, bandages and grafts coated with conditioned media or proteins derived from the media increase the concentration of damaged cells, such as leukocytes and fibroblasts, in the damaged area and induce cell proliferation and differentiation. Resulting in improved wound healing.

  In another embodiment, the conditioned medium can be formulated with a carrier for pharmaceutically acceptable in vivo administration as a vehicle. Further, the medium may be further treated to concentrate or reduce one or more factors or components contained in the medium, for example, enrichment of growth factors using immunoaffinity chromatography or vice versa. Components that are less desirable for any given application as described herein can also be removed.

  Of course, a wound in a specific tissue may require a medium conditioned in that specific tissue. For example, nerve tissue damage requires proteins contained in media conditioned with neuronal cell cultures. A specific product can be induced, in some cases, the conditioned medium can be enriched by immunoaffinity chromatography, or the expression of the protein of interest (eg, NGF) from the specific medium can be enhanced. Features regulated by NGF include cholinergic neurotransmitter function [acetylcholinesterase (AChE) and acetylcholine synthase (ChAT)], neuronal size, and type II NGF receptor expression. However, it is not limited to them. NGF is secreted into conditioned media conditioned by glial cells and other neurons cultured on three-dimensional stromal tissue, which can be used in a composition for nerve healing.

  Endogenous NGF deficiency exacerbates certain human neurodegenerative disorders and apparently renders damaged adult CNS neurons unrenewable. Specifically, when a nerve is damaged, nerve fibers distal to the damage are denatured, causing axonal detachment from the cell body. In the central nervous system, no significant proliferation occurs at the site of injury, and typically damaged neurons die. NGF plays a very important role in the regenerative capacity of adult CNS cholinergic neurons at the cell body level (eg, septal area), intervening tissue cavities (eg, nerve bridges), and innervation regions (eg, hippocampal formation) . In addition, NGF can be beneficial in improving cognitive deficits. For example, medium conditioned with glial cells supplies exogenous NGF and other nerve growth factors, and new axons extend from the cut end of damaged nerves (growth cone development) Can be extended to the original site.

  Furthermore, brain and spinal cord injury is often accompanied by a glial response to concomitant axonal degeneration, resulting in scar tissue. This scar tissue was initially thought to be a physical barrier to nerve development, but what is crucial is the presence or absence of neurotrophic factors in the extraneuronal environment. Astrocytes are thought to be able to synthesize laminin in response to injury (laminin is also found in conditioned media as described in more detail in section 5.8.2 relating to the extracellular matrix. ) Collagen and fibronectin, and in particular laminin, have been shown to promote neurite growth from cultured neurons or neural explants in vitro. These extracellular matrix proteins are thought to provide an adhesive underlayer that promotes forward movement of the growth cone and axonal elongation. Therefore, the presence of neurotrophic factors and supporting substratum is necessary to achieve nerve regeneration. This is because regeneration requires that the neuronal cell body can develop an appropriate biosynthetic response and that the environment surrounding the injury site can support axonal extension and eventual functional recombination. Because it is. Medium conditioned with cells of the nervous system such as astrocytes and glial cells contains neurotrophic factors, nerve growth factors and extracellular matrix proteins necessary for nerve regeneration in brain and spinal cord injury. Thus, in one embodiment, the conditioned medium is formulated for the treatment of such damage.

  In another embodiment, the treatment of skin, bone, liver, pancreas, cartilage and other specific tissues is treated with a medium conditioned with their respective specific cell types, preferably three-dimensional cultures. Can do. Such acclimation provides a conditioned medium containing extracellular proteins and other metabolites of that tissue type that are useful and characteristic for the treatment of wounds of their respective tissue type.

  Cell conditioned media can also be added to the devices used in periodontal surgery to promote uniform tissue repair, or provide surgical void filling to provide biodegradable contact lenses, corneal shields or bone grafts. It can be added to provide an agent, to promote soft tissue augmentation (especially in the skin for the purpose of reducing skin wrinkles) and to enhance the urinary sphincter for the purpose of regulating incontinence is there.

  In another embodiment, the composition can be lyophilized and added as a wound filler (e.g., filling a hole made by an implantable hair plug) or an existing wound filling It can be added to the composition to promote wound healing. In another embodiment, the media can be conditioned with engineered cells to increase the concentration of wound healing proteins in the media. For example, the cells can be made to express a gene product such as any of the growth factors described above.

5.8.2. Repair and correction of congenital malformations, acquired defects and cosmetic defects The medium composition can also be used to repair and correct various congenital and acquired malformations, as well as cosmetic defects that have been invaded and infiltrated. it can. For example, the composition may be added in any form, may be used as a hydrogel, injection, cream, ointment, and further added to eyeshadows, pancakes, makeup products, compact or other cosmetics The skin may be locally reinforced.

  In another embodiment, the topical medium is used to restore and / or prevent sputum and many harmful effects caused by exposure to, for example, UV light, various contaminants and normal aging. Application or application by any known method such as injection, oral, etc. is made.

  Furthermore, in another embodiment, the culture medium of the present invention is used to reduce cellular aging and suppress the activity of factors that cause skin cancer. 7.1. The conditioned medium has antioxidant activity 7.1. It is shown in the section. Again, the application to the mammal may be topical or may be applied by any known method such as injection or oral. Applicants have noticed that human keratinocytes exposed to Applicants' conditioned medium showed a statistically significant (p <0.003) reduction in intracellular oxidation of approximately 50%. .

  Thus, in addition to inducing epithelial and dermal cell proliferation and collagen secretion in vitro, the conditioned medium of the present invention has potent antioxidant activity (FIG. 5). Also, these factors were relatively stable, and the content of TGF β1, VEGF and collagen was stable after storage for 21 days at 37 ° C. and pH 7.4 and 5.5. The solution stored at −20 ° C. for 2 years maintained stable TGF β1 and VEGF levels.

  This sterile enriched nutrient solution is a readily available in large quantity, biologically made medicinal cosmetics that can be used to supplement various levels of growth factors and matrix molecules in human skin, hair and nails. It may be useful as an additive for skin, cosmetic and dermatological products. The product is used with alpha hydroxy acid exfoliants to potentially optimize the penetration of growth factors and other biological components into the skin, and chemically to promote healing and reduce inflammation It is envisaged to be used with a release agent.

Instead of using silicone or other products, the regular conditioned medium may be formulated to eliminate wrinkles, eyebrows, and other skin conditions. Conditioned medium, for example (see 5.8.1. Section Table 3) VEGF, HGF, IL-6 , IL-8, growth factors and inflammatory mediators such as G-CSF and TFGbeta _1, as well as type I and Type III collagen, fibronectin, tenascin, glycosaminoglycan, acidic and basic FGF, TGF-α and TGF-β, KGF, versican, decorin, betaglycens, syndean and other secreted humans Includes extracellular matrix proteins such as skin matrix proteins, which are useful for repairing physical malformations and cosmetic defects. 6.3. As detailed in the section, Applicants have examined the effect of three-dimensional conditioned media on the preparation and composition of three-dimensional tissues and the tissue cultured in the presence of serum-free medium, medium or three-dimensional conditioned medium. It should be noted that the amount of collagen secreted into the extracellular matrix was measured. As shown in FIG. 4, the conditioned medium of the present invention significantly increased tissue collagen accumulation in vitro. The effect of three-dimensional conditioned medium on the preparation and composition of three-dimensional tissues measures the amount of collagen secreted into the extracellular matrix of tissues cultured in the presence of serum-free medium, medium or three-dimensional conditioned medium It was investigated by. Of course, the cells used to acclimate the medium may be genetically engineered to express such proteins at high concentrations in the medium.

  The conditioned medium of the present invention can be formulated into an injectable preparation. Alternatively, the product derived from the conditioned medium may be formulated. For example, biologically active substances, such as proteins and drugs, can be mixed into the composition to release or control release of these active substances after injection of the composition of the invention. Exemplary biologically active substances can include tissue growth factors such as TGF-β that promote healing and tissue repair at the site of injection. Product purification methods include gel chromatography using a matrix such as SEPHADEX® conditioned medium, metal chelate affinity chromatography using an insoluble matrix such as ion exchange, cross-linked agarose gel, HPLC Examples include, but are not limited to, purification, hydrophobic interaction chromatography. Such techniques are described in great detail in Cell & Tissue Culture: Laboratory Procedures, supra; Sanbrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Lab Press, Cold Spring Hgarbor, NY. Has been.

  In an injectable pharmaceutical embodiment, an aqueous suspension is used, and the formulation of this aqueous suspension typically has a physiological pH (ie, pH around 6.8-7.5). In addition, a local anesthetic such as lidocaine (usually a concentration of about 0.3% by weight) is usually added to reduce local pain during injection. The final formulation typically also includes a liquid lubricant such as maltose, which must be tolerated by the body. Representative lubricant components include glycerol, glycogen, maltose and the like. Organic polymer materials such as polyethylene glycol and hyaluronic acid and non-fibrillar collagen, preferably succinylated collagen, can also act as lubricants. Such lubricants generally improve the injectability, intrudability and dispersibility of the injected biomaterial at the site of injection and reduce the amount of spiking by changing the viscosity of the composition. Used for. The final formulation will, of course, contain the treated cell conditioned medium in a pharmaceutically acceptable carrier.

  The treated conditioned medium is then placed in a syringe or other infusion device in order to accurately place the conditioned medium into the tissue defect site. In the case of formulations for skin augmentation, the term “injectable (or injectable)” means that the formulation has a gauge as low as 25, under normal conditions, under normal pressure and without substantial spiking. It means that it can be supplied from a syringe. Spiking can cause the composition to leak from the syringe without being injected into the tissue. For this precise injection, a thin needle of around 27 gauge (diameter 200μ) or at most 30 gauge (diameter 150μ) is desirable. The maximum particle size that can be extruded from such a needle is a complex function of at least the following: maximum particle size, particle aspect ratio (length: width), particle hardness, particle surface roughness And particles: related factors that affect particle adhesion, the viscoelasticity of the suspending liquid, and the flow rate through the needle. Hard spherical beads suspended in Newtonian fluid are the simplest case, and those in which fibrous or branched particles are suspended in a viscoelastic fluid are considered more complex.

  The steps described above in the method of preparing a secreted human conditioned medium for injection are preferably performed using aseptic material under aseptic conditions. Treated conditioned medium in addition to a pharmaceutically acceptable carrier is injected intradermally or subcutaneously to increase soft tissue and repair or correct congenital malformations, acquired defects or cosmetic defects be able to. Examples of such symptoms are unilateral microfacet, cheek and zygomatic dysplasia, unilateral breast dysplasia, funnel chest, no development of the pectoral muscle (Polish malformation), and cleft palate (as a retropharyngeal implant) Congenital malformations such as palatal pharyngeal insufficiency associated with repair or submucosal cleft palate; depressed scars, subcutaneous atrophy (eg, associated with discoid lupus erythematosus), keratopathic lesions, uncleated eyes Acquired defects (trauma) such as eyeball depression [also superior sulcus syndrome], facial acne scars, linear scleroderma with subcutaneous atrophy, sinus deformity, Romberg disease and unilateral vocal cord paralysis Postoperative, postoperative, post-infection); and cosmetic defects such as eyebrows, deep nasal lip, maple folds around the mouth, depressed cheeks and breast failure. The compositions of the present invention can also be injected into internal tissues such as tissues that define the sphincter of the body to increase such tissues.

  Other tissue types that can be used to condition the medium include, but are not limited to, bone marrow, skin, epithelial cells, and cartilage. However, it should be clearly understood that this three-dimensional culture system can be used with other types of cells and tissues.

  Alternatively, the cell conditioned media of the present invention may be formulated with a polymerizable or crosslinkable hydrogel, as described in the previous section for wound treatment.

5.8.3. Food additive and nutritional supplement The conditioned medium can be used as a food additive or formulated into a nutritional supplement. The conditioned medium of the present invention is rich and diverse in many useful nutrients including essential amino acids, minerals and vitamins that are not found in individual foods or commodity groups. Applicants do not know such balanced food ingredients (other than breast milk) that contain a very wide variety of nutrients, but in specially formulated and expensive liquid formulations that are available to both adults and infants Has been tried. Cell conditioned media and / or products derived from it as an inexpensive source of balanced nutritional supplements for weight loss or to increase the nutritional content of food, especially in third world countries Can be used. The medium is sterile and free from contamination by human pathogens (ie, sterile). The conditioned medium can be concentrated and / or lyophilized, preferably administered as a capsule or tablet for consumption. Alternatively, the composition may be added directly to adult or infant food to increase the nutritional content. This nutrient-rich source can be processed relatively inexpensively, especially for undernourished elderly people, especially children in developing countries with increased mortality due to poor resistance to infection related to malnutrition. Can be invaluable.

  In addition, many trace elements found in this conditioned medium, such as iron and magnesium, are important for mammalian survival and reproduction, and the lack of minimal trace elements is a public health issue. There are concerns that could be a problem above. Ingestion of various essential micronutrients has been suggested to reduce cancer risk by modifying specific stages of carcinogenesis as well as infections. Micronutrients also enhance the production of various cytokines, enhance their phagocytic and cytotoxic effects on invading pathogens, and / or premalignant cells that appear in various living organisms. By destroying, it enhances the functional activity of the immune system and the interaction mechanism of its T and B cells, especially Mos and NK cells. See Chandra, R.K. (ed.) (1988), Nutrition and Immunology; Contemporary Issues in Clinical Nutrition, Alan R. Liss, New York. Therefore, there is a need for a relatively inexpensive source of balanced nutrients. Ideal foods enriched with conditioned medium are bread, cereal, and other cereal products such as pasta and crackers. The medium may also be further processed to concentrate or reduce one or more factors or components contained in the medium, for example, enrichment of growth factors using immunoaffinity chromatography, or vice versa. This includes the removal of less desirable components for any given application as described in this section.

5.8.4. Animal Dietary Supplement The composition can also be used as an animal diet supplement. In one embodiment, the conditioned medium contains bovine serum that is a source of proteins and other factors beneficial to mammals such as cattle and other ruminants (eg, dairy cows, deer, etc.). The medium was screened for pathogens but was free of bovine pathogens and mycoplasma. The conditioned medium of the present invention is preferably obtained from dairy cows raised in the United States so that the potential for pathogens is very low.

5.8.5. Cell culture media compositions can be “reused” to culture cells, particularly cells that are difficult to culture in vitro. Conventional growth media is typically supplemented with many of the factors already present in Applicants' conditioned media. In addition, the conditioned medium also contains factors that promote cell adhesion and growth, such as the extracellular matrix proteins described above. Higher concentrations of fibronectin or collagen are advantageous in promoting cell adhesion to the scaffold or culture surface. Rather than adding these factors to the media, conditioned media may be used to culture the cells and prepare a three-dimensional tissue construct such as Dermagraft®. Applicants have demonstrated that this conditioned medium increases cell proliferation of fibroblasts and keratinocytes (see FIG. 3). Cell debris or other particulate matter, as well as other components that are undesirable for protease, lactic acid and cell growth, can be removed from the medium before it is reused as a cell culture medium. For this application, it may be desirable to use serum in cell conditioned media. Serum also contains adhesion factors such as fibronectin and serum diffusion factors that promote cell adhesion to the support. Such adhesion is necessary for in vitro growth of some cells (but not whole cells). Serum not only provides the substances necessary for cell growth, but may also play a role in stabilizing and detoxifying the culture environment. For example, serum has significant buffering capacity and includes certain protease inhibitors such as α ₁ -antitrypsin and α ₂ -macroglobin. Serum albumin is present at high levels, for example in media containing 10% serum, which may not only act as a non-specific inhibitor of proteolysis, but may be toxic in their free form Can bind to fat-soluble vitamins and steroid hormones. Serum components may also detoxify by binding to heavy metals and reactive organics that may be present in the media components.

5.8.6. Medicinal Use The conditioned medium of the present invention contains a variety of useful pharmaceutical factors and ingredients such as growth factors, regulators, peptide hormones, antibodies, etc., as described throughout the specification, and thus is diverse. Useful for various pharmaceutical applications. Also, products that can be added include, but are not limited to, antibiotics, antiviral agents, antifungal agents, steroids, analgesics, antitumor agents, investigational drugs, or consequently in conditioned media. Any compound that is complementary or synergistic with the agent is included. As described above, the cells are cultured and the medium is collected under aseptic conditions. Moreover, this medium can be tested for pathogens. Sterilization should be performed in a manner that minimizes the effect on the desired biological activity, as described above. The medium can be further processed to concentrate or reduce one or more factors or components contained within the medium. For example, for any given application described above, immunoaffinity chromatography can be used to enrich for growth factors or conversely remove unwanted components. In a preferred embodiment, the formulation is made from media conditioned with a three-dimensional cell construct. Three-dimensional culture produces a number of growth factors and proteins that are secreted into the medium at optimal physiological ratios and concentrations. For example, see Table 2 in Section 5.8.1. This medium therefore provides a unique combination of factors and specific ratios that approximate those found in vivo. Bovine serum is generally not preferred for this use. It is preferred to remove not only cell debris or other specific substances, but also proteases, lactic acid, and other components that may be detrimental to cell growth.

  Conditioned media can be tablets, capsules, skin patches, inhalants, eye drops, nasal drops, ear drops, suppositories, creams, ointments, injections, drugs in the form of hydrogels, and any other known to those skilled in the art Can be formulated into a suitable formulation. For oral administration, the pharmaceutical composition comprises, for example, a binder (eg pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg lactose, microcrystalline cellulose or calcium hydrogenphosphate); a lubricant ( Prepared as usual using pharmaceutically acceptable excipients such as, for example, magnesium stearate, talc or silica); disintegrants (eg potato starch or sodium glycolate starch); or wetting agents (eg sodium lauryl sulfate) For example, in the form of tablets or capsules. The tablets may be coated using methods well known in the art. Liquid preparations for oral administration may be presented in the form of, for example, solutions, syrups or suspensions, or as dry products for constitution with water or other suitable vehicle prior to use. Such liquid formulations include suspending agents (eg, sorbitol syrup cellulose derivatives or hydrogenated edible oils); emulsifiers (eg, lecithin or gum arabic); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); And pharmaceutically acceptable additives such as preservatives (eg methyl or propyl-p-hydroxybenzoic acid or sorbic acid) can be prepared as usual. Moreover, you may make this formulation contain a buffer salt, a flavoring agent, a coloring agent, and a sweetening agent suitably.

  Using standard procedures well known to those skilled in the art, the pharmaceutical formulations of the present invention can be delivered to a patient by various routes. For example, such delivery is site specific, oral, nasal, intravenous, subcutaneous, intradermal, transdermal, intramuscular or intraperitoneal administration. They can also be formulated to function as a controlled sustained release vehicle.

  The therapeutic products contained in the conditioned medium include, but are not limited to, enzymes, hormones, cytokines, antigens, antibodies, clotting factors and regulatory proteins. Therapeutic proteins include, but are not limited to, inflammatory mediators, angiogenic factors, factor VIII, factor IX, erythropoietin (EPO), alpha 1 antitrypsin, calcitonin, glucocerebrosidase, human growth hormone and derivatives, low Density lipoprotein (LDL) and apolipoprotein E, IL-2 receptor and its antagonist, insulin, globin, immunoglobulin, catalytic antibody, interleukin (IL), insulin-like growth factor, superoxide dismutase, immune responder modification Factors, BMP (bone morphogenetic protein), parathyroid hormone and interferon, nerve growth factor, tissue plasminogen activator, and colony stimulating factor (CSF). Of course, the medium can be further processed to concentrate or reduce one or more factors or components contained within the medium. For example, for any given application described above, immunoaffinity chromatography can be used to enrich for growth factors or vice versa to remove unwanted components.

  By testing the activity of a particular factor or group of factors using assays commonly used by those skilled in the art, the level of biological activity (e.g., therapeutically effective) by an attached molecule or encapsulated molecule It can be confirmed that the active activity is retained.

  Thus, using the cell conditioned medium of the present invention and products derived therefrom, for example, insulin in the treatment of diabetes, nerve growth factor for the treatment of Alzheimer's disease, factor VIII for the treatment of hemophilia and Other coagulation factors, dopamine for the treatment of Parkinson's disease, enkephalins via adrenal chromaffin cells for the treatment of chronic pain, dystrophin for the treatment of muscular dystrophy, and human growth hormone for the treatment of growth disorders can be provided.

  The dosage of such therapeutic protein agents is well known to those skilled in the art and can be found in the following medical review books: PHYSICIANS DESK REFERENCE, Medical Economics Data Publishers; REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co .; GOODMAN & GILMAN THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw Hill Publ., THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers.

  Any of the therapeutically effective doses of the above agents or agents can be routinely determined using techniques well known to those skilled in the art. A “therapeutically effective” dose refers to that amount of the compound sufficient to effect a reduction in at least one symptom of the process and / or disease to be treated.

Drug toxicity and therapeutic effects can be determined by standard pharmaceutical techniques in cell cultures or experimental animals. For example, determine the LD ₅₀ (lethal dose to 50% of the population) and ED ₅₀ (therapeutically effective dose to 50% of the population). The ratio of toxic to therapeutic dose is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. When using compounds that exhibit toxic side effects, delivery systems that target sites of tissue affected by these compounds to reduce side effects by minimizing potential damage to uninfected cells Care should be taken to design

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Circulating plasma concentrations are within the range that includes the IC ₅₀ determined by cell culture (ie, the concentration of the test compound that achieves half-maximal symptom suppression). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

  In addition, cells and tissues can be genetically manipulated to enhance the expression of desired products such as insulin and / or target the gene products listed above, such as ribozymes, antisense molecules and triple helices. Thus, nucleotide sequences and / or partial molecules that appear to have an inhibitory effect on targeted gene expression and / or activity can be expressed. This may be advantageous when culturing tissues in which specific stromal cells play a specific structural / functional role, such as glial cells of neural tissue, Kupffer cells of the liver, etc. It is done.

5.8.7. Promoting hair growth For example, human hair papilla cells can be used to conditioned the medium. Preferably, the medium conditioned by such cells is grown in three dimensions. Hair papillae cells are a type of mesenchymal stem cell that plays a central role in hair formation, growth and recovery (Matsuzaki et al., Wound Repair Regen. 6: 524-530 (1988)). The conditioned medium is preferably concentrated and applied as a topical formulation. A conditioned medium composition is formulated for topical application using an agent that facilitates penetration of the compound into the skin, such as DMSO, and applied in a topical application method to stimulate hair growth.

  The composition of the present invention, when applied topically, promotes or restores hair growth by providing growth factors and other factors that increase the migration of epithelial cells to the hair follicle. In addition to the growth factors present in the conditioned medium, other compounds such as minoxidil and antibiotics can be used. During hair growth there is a reduction in blood supply during catagen (the transitional phase between the growth and resting phases of the hair follicle) and telogen (resting). For use during these phases of hair growth using assays known in the art, including models of stump-tailed macaque monkeys for juvenile hair loss Biologically active molecules obtained from cell conditioned media can be determined and optimized. See for example: Brigham, Pa, A. Cappas, and H. Uno, The Stumptailed Macaque as a Model For Androgenetic Alopecia: Effects of Topical Minoxidil Analyzed by Use of the Folliculogram, Clin Dermatol, 1988, 6 ( 4): p.177-87; Diani, ARand CJMills, Immunocytochemical Localization of Androgen Receptors in the Scalp of the Stumptail Macaque Monkey, a Model of Androgenic Alopecia, J Invest Dermatol, 1994, 102 (4): p.511 -4; Holland, JM, Animal Models of Alopecia, Clin Dermatol, 1988, 6 (4): p.159-162; Pan, HJ et al., Evaluation of RU58841 as an Anti-Androgen in Prostate PC3 Cells and a Topical Anti- Alopecia Agent in the Bald Scalp of Stumptailed Macaques, Endocrine, 1998, 9 (1): p. 39-43; Rittmaster, RS et al., The Effects of N, N-diethyl-4-methyl-3-oxo-4-aza -5alpha-androstane-17 beta-carboxamide, a 5 alpha-reductase Inhibitor and Antiandrogen, on the Development of Baldness in the Stumptail Macaque, J.Clin Endocrinol Metab, 1987,65 (1): p.188-93 See the full text of each Incorporate Te). Other models include those that measure the difference in hair follicle growth from the hair loss and follicles cultured from the hair part of the newborn rat model and the alopecia areata rat model. See: Neste, DV, The Growth of Human hair in Nude Mice, Dermatol Clin., 1996, 14 (4): p.609-17; McElwee, KJ, EMSpiers, and RFOliver, In Vivo Depletion of CD8 + T Cells Restores Hair Growth in the DEBR Model for Alopecia Areata, Br J Dermatol, 1996,135 (2): p.211-7; Hussein, AM, Protection Against Cytosine Arabinowide-Induced Alopecia by Minoxidil in a Rat Animal Model, Int J Dermatol, 1995, 34 (7): p. 470-3; Oliver, RF et al., The DEBR Rat Model for Alopecia Areata, J Invest Dermatol, 1991, 96 (5): p. 978; Michie, HJ Et al., Immunobiological Studies on the Alopecic (DEBER) Rat, Br J Dermatol, 1990, 123 (5): p.557-67 (the full text of each of which is incorporated by reference).

6). Example
6.1. Conditioning the medium
Human skin fibroblasts were seeded on the substrate of the device described in the '766 patent and described in detail in Sections 5.3, 5.4 and 5.6 above. The substrate is present in the case, designed to promote three-dimensional tissue growth on its surface, and high glucose DMEM (2 mM L-glutamine and 2% at 37 ° C in a humidified 5% CO ₂ atmosphere. Cells were cultured in a closed system cultured in 10% BCS supplemented with 50 mg / ml ascorbic acid). After 10 days, the cell culture was removed and fresh medium was added. Cells were cultured for an additional 4 days as described above. Conditioned media obtained by exposure to cell and tissue culture for 4 days (10-14 days) was then removed from individual compartments and pooled. Distribute this conditioned medium (approximately 5-10 liters / pool) into 200 ml aliquots and use pressure concentrators (Amicon, Beverly, Mass.) With a 10,000 MW cut-off filter for an additional 10-20 times. Concentrated to The resulting 10-20 ml concentrated conditioned medium was distributed into 1 ml aliquots and frozen at -20 ° C for analysis. As a result of adding 10 × conditioned medium as a 10% (vol / vol) solution to the standard medium, a 1 × concentration conditioned medium results. Similarly, as a result of adding 10 × medium (that is, standard medium) or 10 × serum-free medium (standard medium without serum) as a 10% (vol / vol) solution to the standard medium, 1 × concentration of “medium” or These become “serum-free media” which are then used as controls.

6.2. Growth activity of three-dimensional conditioned medium
6.2.1. Exposure of fibroblasts and keratinocytes to conditioned medium The conditioned medium of Section 6.1 was tested for its ability to promote the growth of human fibroblasts and keratinocytes. High glucose supplemented with human fibroblasts or human basal keratinocytes in a 96-well plate (approximately 5,000 cells / well) and supplemented with 1X final concentration serum-free medium or three-dimensional conditioned medium as described in section 6.1 above Cultured in DMEM (10% BCS supplemented with 2 mM L-glutamine and 1 × antibiotic / antimycotic). Cultures were maintained at 37 ° C. for 3 days in a humidified 5% CO ₂ atmosphere.

6.2.2. Cell proliferation Measure cell proliferation using a commercially available fluorescence-based dye assay (CyQuant Cell Proliferation Assay Kit, Molecular Probes, Eugene, Or) that measures total nucleic acid content as part of cell proliferation assessment did. All assays were performed according to manufacturer's instructions. The medium was removed by blotting and the cells were lysed using a lysis buffer containing green fluorescent dye, CyQuant GR dye. This dye exhibits strong fluorescence enhancement when bound to cellular nucleic acid, the amount of fluorescence being proportional to the amount of nucleic acid present in the sample. Samples were incubated for 5 minutes in the absence of light and the fluorescence of the sample was measured using a microtiter plate reader with appropriate filters for about 480 nm excitation and about 520 nm emission maximum. The amount of nucleic acid in each sample was calculated by comparing the amount of fluorescence observed in each well against a standard curve obtained using a known concentration of small bovine thymus DNA as a standard.

  As shown in FIG. 3, cells cultured in medium containing conditioned medium resulted in increased cell proliferation of both fibroblasts and keratinocytes when compared to the two controls.

6.3. Regulation of collagen deposition in tissues by three-dimensional conditioned medium.
6.3.1. Three-dimensional effects on the preparation and composition of three-dimensional tissues by measuring the amount of collagen secreted into the extracellular matrix of tissues cultured in the presence of serum-free medium, medium or three-dimensional conditioned medium for wound treatment The effect of conditioned medium was tested.

Nylon scaffold was laser cut into 11 mm x 11 mm squares, washed in 0.5 M acetic acid, washed thoroughly in FBS, seeded with 8F passage 12F clinical fibroblasts (approximately 38,000 / cm ² ) . DMEM (10% BCS supplemented with 2 mM L-glutamine and 1 × antibiotic / antimycotic) supplemented with 1 × final concentration of serum-free medium, medium or 3D conditioned medium as described in section 6.1 above The culture was grown in 1 ml with the addition of 50 mg / ml ascorbic acid per culture. Copper sulfate was added to a final concentration of 2.5 ng / ml and maintained at high oxygen (40%, about twice that in air) with a standard incubator regulated gas supply. Cultures (n = 3 or higher) were maintained at 37 ° C. for 10 days in a humidified 5% CO ₂ atmosphere. A control without ascorbic acid was also added.

6.3.2. Isolation of collagen From 3D tissue culture grown in the presence of the above 1X final concentration serum-free medium, medium or standard medium supplemented with 3D conditioned medium, collagen is isolated until almost homogeneous Purified. Purified collagen samples are electrophoresed on a gradient SDS-polyacrylamide gel, individual protein bands are visualized with Coomassie Blue, and the amount of alpha, beta and gamma bands specific for collagen compared to total protein (below) Was used to assess the purity of the final sample preparation. As a result of the purification method, similar patterns were generated in all samples.

  Samples were washed in PBS and then with sterile water followed by 2-6 hours in 0.5 M acetic acid. Samples were then digested overnight at 4 ° C. in 1 mg / ml pepsin (Worthington, Inc.) in 0.012 N HCl. Samples were clarified by centrifugation at 13000 rpm at 4 ° C. After the addition of 5 M NaCl to a final concentration of 0.7 M, collagen was precipitated at 4 ° C. for 30-60 minutes. Precipitated collagen was separated by centrifugation at 13000 rpm for 30-60 minutes at 4 ° C. and resuspended in 0.012 N HCl.

6.3.3. Analysis Total protein was measured using a commercially available colorimetric assay kit (Pierce, Inc. BCA assay kit). The assay was performed according to the manufacturer's instructions. Bovine skin collagen (InVitrogen, Carlsbad, CA; Cohesion Technologies, Inc., Palo Alto, Calif.) Was used as a standard for quantifying total protein.

  Samples were then subjected to SDS-PAGE analysis by electrophoresis on a 3-8% gradient gel. The isolated collagen sample was then heated to 95 ° C. in reducing sample buffer. The gel was stained with Coomassie blue to remove the dye and scanned with a computer for visualization.

  As shown in FIG. 4, a statistically significant (p <0.05) increase in collagen deposition of about 50% is noted for 3D cultures treated with 3D conditioned media, compared to any control. It was done. Its activity could not be obtained from the presence of medium or serum alone.

  There are numerous uses for such enhanced collagen deposition in vivo. These can promote wound healing, treatment of wrinkles and contours that appear with bruising, and matrix deposition around bone ridges that are prone to pressure ulcers in paralyzed or bedridden patients included.

7). Antioxidant activity
7.1. Exposure of skin cells to conditioned media Antioxidants have demonstrated the ability to recover / inhibit cell aging and malignant neoplasms. The antioxidant activity of the three-dimensional living medium was measured on human skin cells. At 37 ° C in a 5% CO ₂ atmosphere humidified in a Petri dish in the presence of MCDB153 medium (KGM, Clonetics, Inc.) supplemented with 1x three-dimensional living medium, medium or serum-free medium control Human basal epidermal keratinocytes (˜100,000 / well) were cultured for 3 days. The medium was removed from each sample by incubating in the presence of trypsin, separating the cells from the dish (Sambrook et al., 1989, Molecular Cloning:. A Laboratory Manual, 2 nd Ed, Cold Spring Harbor Lab Press, Cold Spring Harbor, NY) and then centrifuged.

7.2. FACS analysis Separated cells were incubated for 30 minutes at 37 ° C in the presence of 1 mM dihydrorhodamine-1,2,3 (Molecular Probes, Eugene, Or), then Becton-Dickinson (Franklin Lakes, New Jersey) FACSCAN instrument Were analyzed by FACS (fluorescence activated cell sorting) according to the manufacturer's instructions. The amount of intracellular oxidation of reduced dihydrorhodamine dye is directly proportional to the amount of detectable intracellular fluorescence of the oxidation state of the dye.

7.3. Results Human keratinocytes exposed to the cell conditioned media of the present invention were statistically significant (p <0.0003) for intracellular oxidation compared to identical cells incubated in the presence of serum-free or serum-containing media. ) 50% decrease (see Figure 5). The observed differences are not due to serum factors or ascorbic acid present in the control samples. Thus, topical application of media conditioned by three-dimensional culture can be useful as a medicated cosmetic article useful for treating or ameliorating the effects of aging cells.

8). An occlusive patch test to evaluate the in vivo effects of biotechnologically prepared medicated cosmetic nutrient solutions on human skin
8.1. Six healthy adult women (30-60 years old) who obtained experimental design consent were enrolled. Exclusion criteria include hypersensitivity to proteins, skin diseases, skin damage at or near the test site, diabetes, kidney, heart disease or immune disease, anti-inflammatory agents, immunosuppressive agents, antihistamines or topical agents or cosmetics. Use, and pregnancy are included. In order to minimize position or sequence bias, the test substance was applied to the test site (2 sites, 3.8 cm ² ) of the right or left upper arm of each subject in a rotation scheme. Site 1 received a control vehicle and Site 2 received a treatment (ie conditioned medium). The occlusive patch is a Webril non-woven cotton pad containing 0.2 ml of either vehicle or treatment. The patch was covered and held with 3M occlusive, plastic low allergen tape. An obstructive patch was placed on the forearm of three subjects every day for 5 consecutive times in a 24-hour period. The other three subjects received occlusive patches every day for 12 consecutive times in a 24-hour period (treatment continued). The day after the last patch was applied, 2-mm tissue was taken from each site. This protocol is approved by the IRB for the research institute, California Skin Research Institute (San Diego, Ca.), and is in accordance with CFR Title 21, Part 50 and 56.

8.2. Evaluation The overall observation was graded for gloss, peel, crust, cracks, pigmentation enhancement and pigmentation reduction. Using a five-point scale, the stimulus is visually scored, erythema, edema, papules and vesicles (> 25% patch site), and recognizable reactions (<25% patch site), ie infiltration, diffusion And blister reactions with or without hardening were numerically graded. H & E histological evaluation by an authorized pathologist includes biologically active epidermal thickness, epidermal hyperplasia (acanthosis), granular cell layer thickness, inflammatory infiltration, cell division, collagen and elastic fibers Parameters related to the appearance, as well as vasculature.

8.3. result
For 3 subjects who received 5 days of treatment, no adverse events were induced by conditioned media or controls. Average daily stimulation scores for nutrient solution (0.3) and control (0.2) often show no visual response or erythema at both sites, or often show slight fusion or spotted erythema Was meant. Histological examination (trichrome collagen staining) showed healthy tissue for all measured parameters. Thus, the conditioned medium exhibits efficacy compatible with human skin.

9. Modulation of human endothelial cell behavior The effects of conditioned medium and human matrix produced by matrix-bound fibroblasts on angiogenesis and endothelial cell motility were determined. The conditioned medium was produced through the three-dimensional fibroblast culture described herein (described in Sections 5.3, 5.4 and 5.6) and concentrated (10 ×) or lyophilized. The human extracellular matrix was physically separated from the tissue after production.

9.1. Endothelial tubule formation assay Angiogenesis was assessed using an endothelial tubule formation assay with human umbilical vein endothelial cells (HUVEC). Co-culture of lyophilized conditioned medium and HUVEC results in increased tubule formation compared to negative control (preconditioned medium), 4.9 ± 9.31 mm and 0.00 ± 0.00 mm, and tubule formation in concentrated media Increased to 44.10 ± 1.75 mm, and tubule formation increased to 39.3 ± 5.6 mm in the human extracellular matrix.

9.2. Wound assay The confluent layer of endothelial cells was scratched and the occlusion rate of the resulting “wound” was measured and used to assess cell motility. This “wound” assay was measured as the rate of occlusion in mm / h (millimeters / hour). Endothelial cells treated with lyophilized medium showed a rate of 25.59 ± 12.907 mm / h, and 39.56 ± 15.87 mm / h in concentrated medium. The human extracellular matrix showed no increase in speed compared to the negative control (preconditioned medium), 0.00 mm / h and 27.96 ± 0.01 mm / h for the human extracellular matrix and negative control, respectively.

  Thus, the conditioned medium of the present invention modulates the behavior of human endothelial cells, as explained by changes in angiogenesis (tubule formation assay) and cell motility (assessed using a “wound” assay). There is ability.

  The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described above will become apparent to those skilled in the art from the above and accompanying drawings. Such modifications are intended to fall within the scope of the claims.

  Various references have been cited herein, the entire disclosures of which are incorporated herein by reference.

Claims (15)

A method for producing a pharmaceutical composition comprising:
(a) culturing fibroblasts three-dimensionally in a cell culture medium sufficient to meet the auxotrophy required for in vitro cell growth to form a three-dimensional stromal tissue, here The stromal cells adhere to and wrap around the skeleton of a biocompatible non-viable material that forms a three-dimensional structure,
(b) creating a conditioned medium by culturing the cells in vitro until the cell culture medium contains the desired level of extracellular product;
(c) separating the conditioned medium from the cells used to conditioned the medium; and
(d) combining a conditioned medium with a pharmaceutical carrier;
Including the above method,
The pharmaceutical composition requires it to reduce the harmful effects on keratinocytes in an individual caused by exposure to UV light or pollutants or to reduce the appearance of wrinkles associated with aging of the skin The above method, which is used for topical formulation to the individual.   The method of claim 1, wherein the conditioned medium is recovered from the continuous flow system.   The method according to claim 1 or 2, wherein the conditioned medium is cultured in an aseptic environment.   The cell conditioned medium is in the form of a liquid, solid, lyophilized product, powder, gel or film, or has been processed into a liquid, solid, lyophilized product, powder, gel or film. Item 4. The method according to any one of Items 1 to 3.   5. The method of any one of claims 1-4, further comprising treating the cell conditioned medium to enrich or reduce one or more products contained in the medium.   Protein separation techniques including immunoaffinity chromatography, gel chromatography, ion exchange, metal chelate affinity chromatography, HPLC purification, and hydrophobic interaction chromatography for one or more extracellular products from conditioned media 6. The method of claim 5, comprising reducing or enriching.   6. The method of claim 5, wherein the extracellular product is an extracellular matrix protein.   6. The method of claim 5, wherein the extracellular product is a growth factor, anti-inflammatory mediator, enzyme, cytokine, hormone, antigen, antibody, clotting factor, regulatory factor, angiogenic factor, or anti-angiogenic factor.   9. A method according to any one of claims 1 to 8, wherein the three-dimensional framework comprises a network, sponge or polymerized hydrogel.   The method according to claim 1, wherein the fibroblast comprises a genetically engineered cell.   11. The method of claim 10, wherein the genetically engineered cell has been transfected with a foreign gene under the control of an expression element so that the foreign gene product is expressed and secreted into the conditioned medium.   The method according to any one of claims 1 to 11, wherein the parenchymal cells are cultured on a three-dimensional stromal tissue to form a tissue-specific three-dimensional tissue culture.   The method according to any one of claims 1 to 12, wherein the parenchymal cells are cells of skin, liver, kidney, nerve, pancreas, small intestine, urogenital organ, kidney, spleen, bone, bone marrow, or mucosa.   14. The method according to any one of claims 1 to 13, wherein the parenchymal cell comprises a genetically engineered cell.   15. The method of claim 14, wherein the genetically engineered cell has been transfected with a foreign gene under the control of an expression element so that the foreign gene product is expressed and secreted into the conditioned medium.

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